Functionalized chromophoric polymer dots and bioconjugates thereof

ABSTRACT

The present invention provides, among other aspects, functionalized chromophoric polymer dots comprising a hydrophobic core and a hydrophilic cap, and bioconjugates thereof. Also provided are improved methods for preparing functionalized chromophoric polymer dots. Methods for in vivo imaging and molecular labeling are also disclosed.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 16/209,729, filed on Dec. 4, 2018, which is a continuationapplication of U.S. application Ser. No. 13/508,981, filed on Jul. 18,2012, which is a U.S. National Phase Application under 35 U.S.C. § 371of International Application No. PCT/US2010/056079, filed on Nov. 9,2010, which claims benefit of U.S. Provisional Application No.61/259,611, filed on Nov. 9, 2009, which are expressly incorporatedherein by reference in their entirety for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with US Government support under grant numbersR21AG029574, R21CA147831, and R01NS062725, awarded by the NIH. The USGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Fluorescent probes have played a key role in modern cell biology andmedical diagnostics. Organic small dye molecules are generally used influorescent based techniques such as fluorescence microscopy, flowcytometry, and versatile fluorescent assays and sensors. Historically,common fluorophores were derivatives of fluorescein, rhodamine,coumarin, and cyanine etc. Newer generations of fluorophores such as theAlexa Fluors are generally more photostable. However, for many imagingtasks and ultrasensitive assays, their brightness and photostabilitycannot provide sufficient signal to overcome the background associatedwith various autofluorescence and scattering processes within the cells.Other factors such as blinking and saturated emission rate may also posedifficulties in high-speed and high-throughout fluorescent assays.

Advances in understanding biological systems have relied on applicationsof fluorescence microscopy, flow cytometry, versatile biological assays,and biosensors (Pepperkok, R.; Ellenberg, J. Nat. Rev. Mol. Cell Biol.2006, 7, 690-696; Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.;Tsien, R. Y. Science 2006, 312, 217-224). These experimental approachesmake extensive use of organic dye molecules as probes. But intrinsiclimitations of the conventional dyes, such as low absorptivity and poorphotostability, have posed great difficulties in further developments ofhigh-sensitivity imaging techniques and high-throughout assays(Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.;Nann, T. Nat. Methods 2008, 5, 763-775; Fernandez-Suarez, M.; Ting, A.Y. Nat. Rev. Mol. Cell Biol. 2008, 9, 929-943).

As a result, there has been considerable interest in developing brighterand more photostable fluorescent probes. For example, inorganicsemiconducting quantum dots (Qdots) are under active development and nowcommercially available from Life Technologies (Invitrogen). Qdots areideal probes for multiplexed target detection because of their broadexcitation band and narrow, tunable emission peaks. They exhibitimproved brightness and photostability over conventional organic dyes(Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science1998, 281, 2013-2016; Chan, W. C. W.; Nie, S. M. Science 1998, 281,2016-2018; Wu, X. Y.; Liu, H. J.; Liu, J. Q.; Haley, K. N.; Treadway, J.A.; Larson, J. P.; Ge, N. F.; Peale, F.; Bruchez, M. P. Nat. Biotechnol.2003, 21, 452-452; and Michalet, X.; Pinaud, F. F.; Bentolila, L. A.;Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir,S. S.; Weiss, S. Science 2005, 307, 538-544). However, Qdots are notbright enough for many photon-starved applications because of their lowemission rates, blinking, and a significant fraction of non-fluorescentdots (Yao, J.; Larson, D. R.; Vishwasrao, H. D.; Zipfel, W. R.; Webb, W.W. Proc. Natl. Acad. Sci. USA 2005, 102, 14284-14289). There has beenrecent work to develop non-blinking Qdots (Wang, X. Y.; Ren, X. F.;Kahen, K.; Hahn, M. A.; Rajeswaran, M.; Maccagnano-Zacher, S.; Silcox,J.; Cragg, G. E.; Efros, A. L.; Krauss, T. D. Nature 2009, 459,686-689), but their toxicity, caused by the leaching of heavy metalions, is still a critical concern for in vivo applications.

Quantum dots are inorganic semiconductor nanocrystals of the samematerial (for example CdSe/ZnS dot) with different size in the range ofa few nanometers. They exhibit size tunable emission colors due to thequantum confinement effect. As compared to conventional dye, quantumdots are estimated to be 20 times brighter and 100 times more stable.However, these nanoparticles typically require a thick encapsulationlayer to reach the required levels of water-solubility andbiocompatibility, resulting in particle diameters on the order of 15-30nm for an active fluorophore particle size of only 3-6 nm. Therelatively large size of encapsulated quantum dots can significantlyalter biological function and transport of the biomolecules. Quantumdots show broad band absorption and the major absorption part lies inthe UV region, which is not appropriate for most laser basedapplications. Another critical issue.with quantum dot probes is theirtoxicity due to the leaching of heavy metal Cd²⁺ ions. The energy of UVirradiation is close to that of the covalent chemical bond energy ofCdSe nanocrystals. As a result, the particles can be dissolved, in aprocess known as photolysis, to release toxic cadmium ions into thecellular or subcellular environment. The toxicity issue must becarefully examined before their applications in tumor or vascularimaging can be approved for human clinical purposes. Additionally, thelow emission rates, blinking, and a significant fraction ofnonfluorescent dots also raise potential problems, particularly forsingle molecule/particle imaging applications.

An alternative fluorescent nanoparticle is dye doped latex spheres,which exhibit improved brightness and photostability as compared tosingle fluorescent molecules because of multiple dye molecules perparticle and the protective latex matrix. (Wang, L.; Wang, K. M.;Santra, S.; Zhao, X. J.; Hilliard, L. R.; Smith, J. E.; Wu, J. R.; Tan,W. H. Anal. Chem. 2006, 78, 646-654). However, there are also a numberof limitations with the dye-loaded beads such as limited dye-loadingconcentration (a few percent) due to self-quenching, and the relativelylarge particle size (>30 nm) that would preclude sensing schemesinvolving the use of energy transfer to report analyte concentrations.

Light-emitting polymers have attracted an overwhelming interest sincetheir discovery 20 years ago. These materials combine the easyprocessability and outstanding mechanical characteristics of polymerswith the readily-tailored electrical and optical properties ofsemiconductors, therefore find extensive applications in light-emittingdiodes, field-effect transistors, photovoltaic cells, and otheroptoelectronic devices. Fluorescent polymer dots exhibit extraordinarilyhigh fluorescence brightness under both one-photon and two-photonexcitation (Wu, C.; Szymanski, C.; Cain, Z.; McNeill, J. J. Am. Chem.Soc. 2007, 129, 12904-12905. C. Wu, B. Bull, C. Szymanski, K.Christensen, J. McNeill, ACS Nano 2008, 2, 2415-2423.). The fluorescentpolymer dots possess arguably the highest fluorescence brightness/volumeratios of any nanoparticle to date, owing to a number of favorablecharacteristics of semiconducting polymer molecules, including theirhigh absorption cross sections, high radiative rates, high effectivechromophore density, and minimal levels of aggregation-inducedfluorescence quenching. The use of fluorescent polymer dots asfluorescent probes also confers other useful advantages, such as thelack of heavy metal ions that could leach out into solution. However,for applying these probes in biological imaging or sensing applications,an important problem has yet to be solved, that is, the surfacefunctionalization and bioconjugation.

Therefore, there remains a need to develop fluorescent polymer dots withfunctional groups on the surface that allow for probes to be used inbiological systems. The surface functionalization should maintain orenhance the fluorescence brightness or photostability of the hydrophobicpolymer dots, not change the size and the long-term monodispersity ofthe dots in aqueous environment, allow for further conjugation tobiomolecules of a range of types, prevent or minimize non-specificbinding to other biomolecules, and allow for the polymer dots to beproduced on a commercial scale in a cost-effective manner. The presentinvention meets these and other needs by providing, among other aspect,stable, functionalized chromophoric polymer dots (Pdots) andbioconjugates thereof.

SUMMARY OF THE INVENTION

In one aspect of the present invention relates to a functionalizedchromophoric polymer dot. The functionalized chromophoric polymer dothas a core of chromophoric polymer, and a cap of functionalization agentbearing one or more functional groups.

In one aspect, the present invention provides a functionalizedchromophoric polymer dot having a hydrophobic core and a hydrophiliccap. In one embodiment, the functionalized Pdot comprises a chromophoricpolymer and an amphiphilic molecule, having a hydrophobic moiety and ahydrophilic moiety attached to a reactive functional group, wherein thechromophoric polymer is embedded within the hydrophobic core of the Pdotand; wherein a portion of the amphiphilic molecule is embedded withinthe core of the Pdot and the reactive functional group is located in thehydrophilic cap. In a preferred embodiment, the chromophoric polymer isa semiconducting polymer.

In another aspect, a bioconjugate of the polymer dot is disclosed. Thebioconjugate is formed by the attachment of a biomolecule to one or morefunctional groups of the chromophoric polymer dot. The attachment may bedirect or indirect.

In yet another aspect a method of preparing functionalized chromophoricpolymer dots is disclosed. The method involves the introduction of aprotic solvent into an aprotic solution containing a mixture of achromophoric polymer and a functionalization agent (bearing one or morefunctional groups).

In one aspect, the present invention provides a method for preparing afunctionalized chromophoric polymer dot, the method comprising the stepsof (a) preparing a mixture of a chromophoric polymer and an amphiphilicmolecule attached to a reactive functional group in a non-proticsolvent; (b) injecting all or a portion of the mixture into a solutioncomprising a protic solvent, thereby collapsing the chromophoric polymerand amphiphilic molecule into a nanoparticle; and (c) removing thenon-protic solvent from the mixture formed in step (b), thereby forminga suspension of functionalized chromophoric polymer dots, wherein aportion of the amphiphilic molecule is embedded within the core of thenanoparticle and the reactive functional group is located on the surfaceof the nanoparticle.

In another aspect, the present invention provides a method forconjugating a biological molecule to a functionalized chromophoricpolymer dot, the method comprising incubating a functionalizedchromophoric polymer dot with the biological molecule in a solutioncontaining polyethylene glycol under conditions suitable for conjugatingthe biological molecule to the functionalized chromophoric polymer dot,wherein the presence of polyethylene glycol in the solution reducesnon-specific adsorption of the biological molecule to the surface of thepolymer dot.

In yet another aspect, the present invention provides a method forlabeling a target molecule in a biological sample, the method comprisingcontacting the biological sample with a chromophoric polymer dotconjugated to a targeting moiety that specifically binds the targetmolecule.

In another embodiment, the present invention provides a method for thebioorthogonal labeling of a cellular target, the method comprisingcontacting a cellular target having a first surface-exposed functionalgroup capable of participating in a bioorthogonal chemistry reactionwith a chromophoric polymer dot. In a particular embodiment, thebioorthogonal reaction is a click chemistry reaction that is performedwith a Pdot carrying a reactive functional group capable ofparticipating in such a reaction.

In yet another embodiment, the present invention provides chromophoricpolymer dots having a red-shifted emission peak. In one embodiment, thered-shifted Pdots have a peak emission in the far-red region. In otherembodiments, the red-shifted Pdots have a peak emission in the near-IRregion. In one embodiment, the red-shifted Pdots comprise a PFTBTpolymer. In certain embodiments, the polymer dots comprising a blend oftwo or more different chromophoric polymers. For example, in oneembodiment, the Pdots comprise a blend of PFBT and PFTBT polymers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram for preparing the functionalizedchromophoric polymer dots and their biomolecular conjugates of thepresent invention. Chemical structures of a general chromophoric polymerPFBT and an amphiphilic functionalization polymer PS-PEG-COOH aresketched in the scheme.

FIG. 2 (A) Shows atomic force microscope (AFM) image of functionalizedchromophoric PFBT dots prepared in accordance with the method of thepresent invention. FIG. 2 (B) Shows a particle height histogram takenfrom an AFM image.

FIG. 3 shows the absorption and emission spectra of functionalizedchromophoric PFBT dots prepared in accordance with the method of thepresent invention.

FIG. 4 (A) Shows fluorescence confocal image of SK-BR-3 cancer cellsincubated with primary anti-EpCAM, and then chromophoric PFBT dotsconjugated with secondary anti-mouse IgG antibody. FIG. 4 (B) Showsfluorescence confocal image of SK-BR-3 cancer cells incubated withchromophoric PFBT dots conjugated with secondary anti-mouse IgGantibody. No primary antibody is used in the control FIG. 4 (B).

FIG. 5 (A) Shows fluorescence confocal image of MCF-7 cancer cellsincubated with anti-tubulin biotin primary antibody, and thenchromophoric PFBT dots conjugated with streptavidin. FIG. 5 (B) Showfluorescence confocal image of MCF-7 cancer cells incubated with biotinanti-tubulin primary antibody, and then bare PFBT dots. The PFBT dotswithout streptavidin conjugation are used as a control.

FIG. 6 (A) Shows a generalized CPdot of the invention, the core could bea semiconducting polymer, a non-semiconducting chromophore-containingpolymer (such as dyes, metal complexes, and the like), a semiconductingpolymer with optical inert polymer or inorganic functional materials, ortheir combinations; the cap could be a small molecule bearing functionalgroups, a surfactant bearing functional groups, a lipid bearingfunctional groups, or a polymer bearing functional groups. The cap andthe core could be held together by chemical bonding or by physicalassociation. FIG. 6 (B) Shows a preferred CPdot of the invention thecore could be a semiconducting polymer, a non-semiconductingchromophore-containing polymer (such as dyes, metal complexes, and thelike), a semiconducting polymer with optical inert polymer or inorganicfunctional materials, or their combinations; the cap comprises anamphiphilic polymer which has a hydrophobic moiety and a hydrophilicmoiety. The hydrophobic moiety is embedded in the core by physicalassociation or by chemical bonding and the hydrophilic moiety, whichpossess functional groups for bioconjugation, extends out forbioconjugation.

FIG. 7 (A) Typical AFM image of functionalized PFBT dots. FIG. 7 (B)Histogram of particle height taken on AFM images of functionalized PFBTdots. FIG. 7 (C) An assay using biotin silica beads to verifybioconjugation through EDC-catalyzed covalent coupling. FIG. 7 (D)Absorption and fluorescence spectra of PFBT dot-streptavidinbioconjugates in 1×PBS buffer solution after 6 months of storage, theinset shows photographs of the Pdot-bioconjugate solution under room(left picture) and UV (right picture) illumination.

FIG. 8A-F Single-particle fluorescence images of FIG. 8 (A) PFBT dot,FIG. 8 (B) IgG-Alexa 488, and FIG. 8 (C) Qdot 565, obtained underidentical excitation conditions. Note the color bar for IgG-Alexa andQdot 565 has to be set to a lower value (8000 counts rather than 60,000counts) because they are significantly dimmer than PFBT dots. Scale barrepresents 5 μm. FIG. 8 (D) Signal and background for single Pdots ascompared to single IgG-Alexa 488 and single Qdots, observed underidentical excitation power of 1 mW. FIG. 8 (E) Intensity distributionsof single particle fluorescence for the three probes under theexcitation power of 4 mW. Pdots are ˜30 brighter than either IgG-Alexa488 or Qdots. FIG. 8 (F) Single-particle photobleaching trajectories.Blinking was not observed for PFBT dots (blue), while frequent blinkingwas observed for Qdots (red).

FIG. 9A-B Fluorescence imaging of cell-surface marker (EpCAM) in humanbreast cancer cells labeled with Pdot bioconjugates. FIG. 9 (A) Imagingof live MCF-7 cells incubated sequentially with anti-EpCAM primaryantibody and Pdot-IgG conjugates. The bottom panels show control samplesin which the cells were incubated with Pdot-IgG alone (no primaryantibody). The Nomarski (DIC) images are shown to the right of theconfocal fluorescence images. Scale bar represents 20 μm. FIG. 9 (B)Imaging of live MCF-7 cells incubated sequentially with anti-EpCAMprimary antibody, biotinylated goat anti-mouse IgG secondary antibody,and Pdot-streptavidin conjugates. The bottom panels show control sampleswhere the cells were incubated with anti-EpCAM antibody andPdot-strepavidin (no secondary antibody). The Nomarski (DIC) images areshown to the right of the confocal fluorescence images. Scale barrepresents 20 μm.

FIG. 10A-D Flow-through detection of fluorescently labeled cancer cells.FIG. 10 (A) Fluorescence intensity distributions obtained by flowingPdot-streptavidin labeled MCF-7 cells through a microfluidic flowcytometer; laser excitation was varied from 0.1 to 0.5 to 1 mW. FIG. 10(B) Fluorescence intensity distributions for Qdot 565-streptavidinlabeled MCF-7 cells obtained under identical experimental conditions asthose used in FIG. 10 (A). FIG. 10 (C) Comparison of averagefluorescence brightness obtained using the microfluidic flow cytometerfor cells labeled with Pdot-streptavidin and Qdot-streptavidin. FIG. 10(D) The same experiment and comparison as described in FIG. 10 (A-C) wascarried using Pdot-IgG and Alexa 488-IgG.

FIG. 11 (A) Fluorescence intensity distributions obtained by flowingPdot-IgG-labeled MCF-7 cells through a microfluidic flow cytometer;laser excitation was varied from 0.1 to 0.5 to 1 mW. FIG. 11 (B)Fluorescence intensity distributions for Alexa 488-IgG-labeled MCF-7cells obtained under identical experimental conditions as those used inFIG. 11 (A).

FIG. 12 (A) Fluorescence images for Qdot 565-streptavidin-labeled MCF-7cells obtained on a low numerical aperture wide-field microscope. FIG.12 (B) Fluorescence images for Pdot-streptavidin-labeled MCF-7 cellsobtained under identical conditions as those used in FIG. 12 (A). FIG.12 (C) Fluorescence intensity distributions of Pdot-labeled cellscompared to Qdot-labeled ones.

FIG. 13 Fluorescence decay lifetime (0.6 ns) of PFBT dots measured by aTCSPC setup. The blue line represents experimental data, and the greenline is fitting curve obtained employing an iterative deconvolutionmethod. Residual is shown below the curves (red line).

FIG. 14 Functionalization and conjugation of fluorescent semiconductingpolymer dots for bioorthogonal labeling via click chemistry. A copolymerPSMA was co-condensed with a fluorescent semiconducting polymer, such asPFBT (depicted as green string), thereby forming Pdots with surfacecarboxyl groups. The carboxyl groups enabled further surfaceconjugations to functional molecules for copper (I)-catalyzed clickreaction. The functionalized Pdots were selectively targeted againstnewly synthesized proteins or glycoproteins (blue string) in mammaliancells that were metabolically labeled with bioorthogonal chemicalreporters.

FIG. 15 Absorption and fluorescence spectra of carboxyl functionalizedPFBT dots.

FIG. 16 The fluorescence intensity of PFBT Pdots in HEPES buffer with pHranging from 4 to 9. No obvious change was observed.

FIG. 17A-E Characterization of functionalized Pdots. FIG. 17 (a)Fluorescence photographs of Pdots versus Qdots in the presence of copper(I) under UV illumination. FIG. 17 (b) Migration bands of Pdots withdifferent surface functional groups. FIG. 17 (c) Hydrodynamic diameterof carboxyl functionalized Pdots measured by dynamic light scattering;inset shows a typical TEM image of functionalized Pdots. FIG. 17 (d) Afluorescent assay using alkyne-Alexa 594 dye to verify successfulfunctionalization of Pdots with azido groups. FIG. 17 (e)Single-particle fluorescence images of alkyne-silica nanoparticlescoupled to azido-Pdots by click reaction. Scale bar represents 50 μm.

FIG. 18A-H Fluorescence imaging of newly synthesized proteins in theAHA-treated MCF-7 cells tagged with Pdot-alkyne probes. FIG. 18 (a-d)Positive Pdot labeling in the presence of copper (I). FIG. 18 (e-h) Pdotlabeling in the control sample was carried out under identicalconditions as in FIG. 18 (a-d) but in the absence of the reducing agent(sodium ascorbate) that generates copper (I) from copper (II). The toprow shows fluorescence images; green fluorescence is from Pdots and bluefluorescence is from the nuclear stain Hoechst 34580. The bottom rowshows Nomarski (DIC) and combined DIC and fluorescence images. Scale barrepresents 20 μm.

FIG. 19 Copper (I)-catalyzed Pdot-alkyne tagging was performed underidentical conditions as those in FIG. 18a-18d but in cells not exposedto AHA. In this control, cell labeling was not observed. The top rowshows fluorescence images; blue fluorescence was from the nuclear stainHoechst 34580; no fluorescence from Pdots was observed (top rightpanel). The bottom row shows Nomarski (DIC) (lower left panel) andcombined DIC and fluorescence (lower right panel) images. Scale barrepresents 20 μm.

FIG. 20A-H Fluorescence imaging of newly synthesized proteins in MCF-7cells tagged with Pdot-azide. FIG. 20 (a-d) Copper (I)-catalyzedpositive Pdot labeling in the HPG-treated cells. FIG. 20 (e-h) Pdotlabeling in the control sample was carried out under identicalconditions as in FIG. 20 (a-d) but in cells not exposed to HPG. The toprow shows fluorescence images; green fluorescence is from Pdots and bluefluorescence is from the nuclear stain Hoechst 34580. The bottom rowshows Nomarski (DIC) and combined DIC and fluorescence images. Scale barrepresents 20 μm.

FIG. 21A-H Fluorescence imaging of glycoproteins in GalNAz-treated MCF-7cells tagged with Pdot-alkyne probes. FIG. 21 (a-d) Positive Pdotlabeling in the presence of copper (I). FIG. 21 (e-h) Pdot labeling inthe control sample was carried out under identical conditions as in FIG.21 (a-d) but in the absence of copper (I). The top row showsfluorescence images; green fluorescence is from Pdots and bluefluorescence is from the nuclear stain Hoechst 34580. The bottom rowshows Nomarski (DIC) and combined DIC and fluorescence images. Scale barrepresents 20 μm.

FIG. 22 Three schemes for bioconjugation via click chemistry.

FIG. 23 Multicolor semiconducting polymer dots based on polyfluorenepolymers. While the emission color can be tuned to deep-red region, thedominant absorption is in the UV region to maintain high fluorescencequantum yields. The dominant UV absorption is due to the majority offluorene unit. The left shows chemical structures of these polymers.

FIG. 24 A light-harvesting polymer PFBT, and a red-emitting polymerPF-0.1TBT, and a functional copolymer PSMA was co-condensed to formhighly fluorescent PBdots with surface carboxyl groups. The carboxylgroups enabled further surface conjugations to a tumor-specific peptideligand CTX.

FIG. 25 (A) Chemical structures of PFBT and PF-0.1TBT polymers. FIG. 25(B) Concentration dependent absorption spectra (left) and emissionspectra (right) of PBdots containing a light-harvesting PFBT donorpolymer and a red-emitting PF-0.1TBT acceptor polymer. At a blendingratio of 0.6 (PF-0.1TBT to PFBT), the PBdots show broad absorption invisible region and efficient deep-red emission from PFTBT. The top showschemical structures of PFBT and PF-0.1TBT. FIG. 25 (C) Absorbancespectra (left) and fluorescence emission spectra (right) of the quantumyield (QY) standard DCM dye and PBdots. The absorbance of both PBdots inwater and DCM in methanol was adjusted to be 0.1. Fluorescence spectrawere taken under identical spectrometer conditions.

FIG. 26 (a) Absorption and emission spectra of PBdot. The inset showsphotographs of aqueous PBdot solution under room light (left) and UVlight (right) illumination. FIG. 26 (b) Single-particle fluorescenceimage of Qdot 655 nanocrystals. Scale bar, 4 μm. FIG. 26 (c)Single-particle fluorescence image of PBdot. Images were obtained underthe same excitation conditions as those for Qdot 655, but the 90% of theemitted light was blocked by a neutral density filter (OD=1) to avoiddetector saturation. Scale bar, 4 μm. FIG. 26 (d) Intensitydistributions of single particle fluorescence. PBdots are ˜15 timesbrighter than Qdot 655 probes. FIG. 26 (e) TEM image of carboxylfunctionalized PBdots. Scale bar, 100 nm. FIG. 26 (f) Gelelectrophoresis of functionalized and bioconjugated PBdots. PBdotsconjugated with different biomolecules showed shifted migration bands inan agarose gel, indicating successful functionalization and biomolecularconjugation.

FIG. 27 Absorption spectra (left) and emission spectra (right) of PBdots containing a light-harvesting PFPV donor polymer, and twored-emitting polymers PF-0.1DHTBT, PF-0.1 TBT, respectively. The leftshows the chemical structures of the polymers.

FIG. 28 Fluorescence decay lifetime (3.5 ns) of PBdots measured by atime correlated single photon counting instrument (TCSPC). The blackline represents experimental data, and the red line is the fittingcurve.

FIG. 29 Particle size distribution of PFBT co PFTBT PB dots measured bydynamic light scattering (Malvern Zetasizer NanoZS).

FIG. 30 Fluorescence intensity of PFBT co PFTBT PB dots in HEPES bufferwith pH ranging from 4 to 9. No obvious change was observed.

FIG. 31 (a) Confocal imaging of live MCF-7 cells incubated sequentiallywith anti-EpCAM primary antibody, biotinylated goat anti-mouse IgGsecondary antibody, and PBdot-streptavidin conjugates. FIG. 31 (b)Negative control for PBdot cell labeling where no biotinylated secondaryantibody was used. For both a and b are shown phase contrast images(left) and the combined fluorescence images (right). Red fluorescence isfrom PBdots and blue fluorescence is from the nuclear stain Hoechst34580. Scale bar, 40 μm. FIG. 31 (c) Photobleaching curves extractedfrom confocal fluorescence images obtained under continuous laserscanning for 20 minutes. FIG. 31 (d) Fluorescence stability of PBdotstowards biologically relevant ions and ROS in MilliQ water. Qdot 655nanocrystals were shown for a reference. The concentration for eachmetal ion is 500 μM. For ROS stability test, the H₂O₂ and chlorineconcentration is 1×10⁻³, and 4×10⁻⁵, respectively. To preventaggregation, the solution contained PEG.

FIG. 32A-C Schematic showing the sensing of Cu²⁺ and Fe²⁺ using PS-COOHfunctionalized PFBT Pdots. The PFBT Pdots were first functionalized withPS-COOH, which served as a chelating group FIG. 32 (A). The aggregationand quenching of Pdots was induced by adding Cu²⁺ and/or Fe²⁺ FIG. 32(B). The Cu²⁺-induced aggregation could be reversed by introducing EDTAinto solution, while the aggregation of Pdots resulting from binding toFe²⁺ could not be redispersed FIG. 32 (C). The inset in the center showsphotographs of each corresponding solution under a 365 nm lamp.

FIG. 33A-B TEM images of PS-COON co PFBT Pdots before FIG. 33 (A) andafter FIG. 33 (B) the addition of Cu²⁺, which illustrate Cu²⁺ inducedaggregation of the carboxyl functionalized Pdots. The scale bars are 200nm FIG. 33 (C) Dynamic light scattering measurements of Pdots before andafter addition of Cu²⁺, as well as after addition of EDTA. Hydrodynamicdiameter of PFBT co PS-COOH Pdots measured by DLS in water (▪) and inCu²⁺ containing solution (▪), as well as after the addition of EDTA tothe Cu²⁺ containing solution (▪).

FIG. 34 Effects of different ions (20 μM) on the fluorescence intensityof solutions containing PS-COOH co PFBT Pdots and PFBT co PFTBT Pdots.Images on the top show each sample under a 365 nm lamp.

FIG. 35 (A) Effect of various concentrations of Cu²⁺ ions on thefluorescence of solutions containing PS-COOH co PFBT Pdots and PFBT coPFTBT Pdots. Concentrations of Cu²⁺ ranged from 0 to 30 μM: black line:0, red line: 100 nM, pink line: 500 nM, gold line: 1 μM, plum line: 5μM, green line: 10 μM, orange line: 20 μM, blue line: 30 μM. FIG. 35 (B)A plot of the ratio of the 540 nm peak (from PS-COOH co PFBT Pdots) overthe 623 nm peak (from PFBT co PFTBT Pdots) as a function of the Cu²⁺concentration. The red line is a linear fit to the data (R²=0.992). FIG.35 (C) The fluorescence intensity of Pdots after quenching by Cu²⁺ ions(30 μM) can be recovered by the addition of 30 μM EDTA.

FIG. 36 (A) Effect of various concentrations of Fe²⁺ on the fluorescenceof PS-COOH co PFBT Pdots at 540 nm; the solution also contained PFBT coPFTBT Pdots (emission centered at 623 nm) as an internal standard.Concentration of Fe²⁺ from 0 to 40 μM: black line: 0, red line: 10 μM,green line: 15 μM, pink line: 20 μM, orange line: 25 μM, blue line: 40μM. FIG. 36 (B) A plot of the ratio of the 540 nm peak (from PS-COOH coPFBT Pdots) over the 623 nm peak (from PFBT co PFTBT Pdots) as afunction of the Fe²⁺ concentrations. The red line is a linear fit to thedata (R²=0.996).

FIG. 37 Preparation of near-IR (NIR) dye-doped and functionalized CPdots(interchangeably called Pdots). Chromophoric polymer PFBT, amphiphilicpolymer PS-PEG-COOH, and NIR dyes were mixed together in THE andco-precipitated in water under sonication to form NIR dye-doped Pdots.The Pdot matrix can absorb blue light and transfer the energy to thedoped NIR dyes (indicated by green arrows), which then generate strongNIR fluorescence (indicated by red arrows).

FIG. 38A-C Characterization of NIR dye-doped and functionalized CPdots(interchangeably called Pdots). FIG. 38 (A) TEM image of NIR dye-dopedPdots. The diameter of the CPdots was 18 nm. Scale bar: 50 nm. FIG. 38(B) Number average diameter of NIR dye-doped CPdots measured by DLS.FIG. 38 (C) Agarose gel electrophoresis. Bare PFBT dots, Carboxyl PFBTdots and NIR dye-doped CPdots were loaded in an agarose gel containing0.7% of agarose and 0.2% of PEG, and run at a force of 10V/cm in 20 mMHEPES for 15 min. Bare PFBT dots run slower due to lacking of carboxylsurface. Carboxyl PFBT dots and NIR dye-doped CPdots both containcarboxyl groups on their surfaces and run faster.

FIG. 39 Excitation and emission spectra of PFBT dots, NIR dyes, and NIRdye-doped CPdots (interchangeably called Pdots). The upper Figure showsthe excitation spectra of PFBT dots (Black dash line with shadow) andNIR dyes (Dark yellow dash line with shadow), and the emission spectraof PFBT dots (Blue solid line) and NIR dyes (Red solid line). The lowerfigure shows the excitation (Black dash line with shadow) and theemission (Red solid line) of NIR dye-doped CPdots.

FIG. 40A-E Fluorescence properties of NIR dye-doped CPdots(interchangeably called Pdots). FIG. 40 (A) Fluorescence spectra of NIRdye-doped CPdots with different NW dye doping. FIG. 40 (B) Overlapbetween the normalized emission spectrum of PFBT dot (donor) and thenormalized absorption spectrum of NIR dye (acceptor); the calculatedForster radius between the donor and acceptor pair (Ro) is 3.7 nm. FIG.40 (C) Fluorescence lifetime measurement of CPdots. The lifetime of bareCPdots is 2.4 ns (Experimental data: Green dots, Fitting: Blue solidline), and is reduced to 1.2 ns after doping with NIR dyes (Experimentaldata: Red dots, Fitting: Red solid line). Black solid line representsthe internal reference (IRF). FIG. 40 (D) Applied quenching effect ofNIR dyes in Pdot matrix. The fluorescence intensity ratio of theoriginal CPdots without dye doping (F₀) and the NIR dye-doped CPdots (F)is proportional to the concentration of dopants (Hollow dots with errorbar). Data were fit with the Stern-Volmer equation (solid line). FIG. 40(E) Manipulating the NIR emission of NIR dye-doped CPdots by controllingthe dopant concentration. Increase the dye concentration led to adecrease of NIR emission (Red columns). The black column with asteriskrepresents the 546 nm emission of CPdots without dye doping.

FIG. 41A-C Fluorescence enhancement of NIR dyes inside CPdot matrix(interchangeably called Pdots). FIG. 41 (A) Normalized fluorescence peakintensities of free NIR dyes and the doped NIR dyes excited at 450 nm(Blue columns) and at 763 nm (black patterned columns). The fluorescenceof free NIR dyes was measured in THF, others were measured in 20 mMHEPES buffer (pH 7.4). FIG. 41 (B) Fluorescence emissions of fluorescentnanomaterials at same particle concentration. FIG. 41 (C) The peakintensity of fluorescent nanomaterials normalized by the particleconcentration.

FIG. 42A-B Leaking test of NIR dye-doped CPdots. NIR dye leakage can bemonitored by the change of peak ratio between the fluorescence emissionsof 778 nm (acceptor) and 546 nm (donor). FIG. 42 (A) Dye leakage testedat room temperature in 20 mM HEPES buffer (pH 7.4). The NIR emissionkept unchanged in 72 hours (Blue hollow triangle), the acceptor-to-donorratio slightly decreased after 72 hours (Red hollow square). FIG. 42 (B)Dye leakage tested at 37° C. in human plasma. Data measured in plasma(Blue hollow triangle) are comparable to the data in 20 mM HEPES buffer(Red hollow square).

FIG. 43A-B Scheme for preparing CPdots temperature sensors.

FIG. 44A-C TEM and dynamic light scattering of CPdot temperaturesensors. FIG. 44 (A) TEM of CPdot PFPV-RhB. FIG. 44 (B) TEM of CPdotPFBT-RhB. FIG. 44 (C) Dynamic light scattering of CPdot temperaturesensors.

FIG. 45A-B Absorption and emission spectra of CPdot temperature sensors.

FIG. 46A-B Emission spectra of CPdot PFBT-RhB FIG. 46 (A) and PFPV-RhBFIG. 46 (B) sensors at different temperatures.

FIG. 47A-B Intensity-temperature plot and their linear fittings forCPdot temperature sensors.

FIG. 48A-B Intensity ratio vs. temperature and the linear fittings forCPdot temperature sensors.

FIG. 49A-C Schematic showing three routes for the preparation of PPEPdot-based pH sensor. FIG. 49 (A) PS-SH co PPE Pdots in water were firstprepared and then reacted with the isothiocyanate moieties on the FITCmolecules. FIG. 49 (B) FITC was first conjugated to PS-NH₂ polymersthrough the amine-isothiocyanate reaction, and the resultingfluorescein-labeled PS polymers were blended with PPE polymers to formthe PS-NH₂-FITC co PPE Pdots. FIG. 49 (C) PS-SH co PPE Pdots were firstprepared in the same way as FIG. 49 (A), but were subsequently coupledto fluorescein-5-maleimide. PPE:poly(2,5-di(3′,7′-dimethyloctyl)phenylene-1,4-ethynylene; PS:polystyrene polymer; FITC: fluorescein isothiocyanate.

FIG. 50 (A) UV-visible spectra of PPE Pdots (dashed plum line) and FITC(dashed black line) in water; and emission spectra of PS-SH co PPE(solid red line), PS-SH-FITC co PPE (solid blue line), and PS-NH₂-FITCco PPE Pdots in pH=7 HEPES buffer solutions. The inset in theupper-right corner shows the photographs of PS-SH co PPE (left) andPS-SH-FITC co PPE pdot solutions (right) under a 365 nm UV lamp. FIG. 50(B) Transmission electron microscopy images of the CPdot pH sensors.FIG. 50 (C) Comparison of lifetime of CPdots.

FIG. 51A-C Fluorescence spectra of FIG. 51 (A) Pdot(A), FIG. 51 (B)Pdot(B), and FIG. 51 (C) Pdot(C) at different pH, ranging from 5 to 8(black line: pH=8, red line: pH=7.5, blue line: pH=7, green line:pH=6.5, gold line: pH=6, brown line: pH=5.5, pink line: pH=5).Excitation wavelength was 390 nm.

FIG. 52A-B PH sensitivity and reversibility of the three fluoresceinconjugated Pdots. FIG. 52 (A) Ratiometric pH calibration plot of theemission ratio (I_(513 nm)/I_(440 nm)) of Pdot(A) (●), Pdot(B) (▪), andPdot(C) (▴) as a function of pH. The blue, black, and red lines arelinear fit to the data of Pdot(A) (R²=0.995), Pdot(B) (R²=0.991), andPdot(C) (R²=0.994), respectively. The slopes for Pdot(A), Pdot(B), andPdot(C) are 0.37, 0.18, and 0.29, respectively. FIG. 52 (B) Theintensity ratio (I_(513 nm)/I_(440 nm)) of Pdot(C) when the pH wastoggled between 5.0 and 8.0 repeatedly, illustrating the reversibilityand reproducibility of pH sensing.

FIG. 53A-B Illustration of spectral overlap between the emission ofdonor (i.e., PPE) and the absorption of acceptor (i.e., FITC); and theircalculated Forster distance, Ro. FIG. 53 (A) Emission spectrum ofPS-SH-FITC co PPE Pdots (dashed light green line) and excitation spectraof FITC at pH ranging from 5 to 8. The areas under curves were filledwith color for easier observation of spectral overlap. FIG. 53 (B) Thecorresponding Forster distance of PPE-FITC at different pH was plottedbased on the overlap integral as shown in FIG. 53 (A).

FIG. 54A-H Confocal scanning microscopy images of HeLa cells labeled byPPE Pdots FIG. 54 (A-C) and PS-SH-FITC co PPE Pdots FIG. 54 (E-G); andtheir corresponding bright-field images shown in FIG. 54 (D) and FIG. 54(H), respectively. The blue fluorescence shown in FIG. 54 (A) and FIG.54 (E) was produced by integrating the spectral region of Pdots from433-444 nm, while the green fluorescence shown in FIG. 54 (B) and FIG.54 (F) was integrated from 507-518 nm. The images FIG. 54 (C) and FIG.54 (G) are the overlay of blue and green fluorescence.

FIG. 55A-H Confocal microscopy images of HeLa cells labeled by Pdot(B)FIG. 55 (A-C) and Pdot(C) FIG. 55 (E G) at λ_(exc)=405 nm; theircorresponding bright-field images are shown in FIG. 55 (D) and FIG. 55(H), respectively. The blue channel shown in FIG. 55 (A) and FIG. 55 (E)was produced by integrating the spectral region from 433-444 nm, whilethe green channel shown in FIG. 55 (B) and FIG. 55 (F) was from 507-518nm. The images in FIG. 55 (C) and FIG. 55 (G) are the overlay of theblue and green channels. The insets show a magnified view of a singleHeLa cell. The scale bars are 20 μm.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

Semiconducting polymers are attractive materials for variousoptoelectronic applications, including light-emitting diodes,field-effect transistors, and photovoltaic devices (See, for example,Friend, R. H.; Gymer, R. W.; Holmes, A. B.; Burroughes, J. H.; Marks, R.N.; Taliani, C.; Bradley, D. D. C.; Dos Santos, D. A.; Bredas, J. L.;Loglund, M.; Salaneck, W. R. Nature 1999, 397, 121; and Gunes, S.;Neugebauer, H.; Sariciftci, N. S. Chem. Rev. 2007, 107, 1324-1338, thedisclosures of which are herein incorporated by reference in theirentireties for all purposes). Their appeal is based on thereadily-tailored electrical and optical properties of semiconductorscombined with the easy processability of polymers. Water-solublesemiconducting polymers have also been demonstrated as highly sensitivebiosensors and chemical sensors (see, for example, Chen, L.; McBranch,D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl.Acad. Sci. USA 1999, 96, 12287-12292; Fan, C. H.; Wang, S.; Hong, J. W.;Bazan, G. C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. USAUSA 2003, 100, 6297-6301; and Thomas, S. W.; Joly, G. D.; Swager, T. M.Chem. Rev. 2007, 107, 1339-1386, the disclosures of which are hereinincorporated by reference in their entireties for all purposes).

Since the early demonstration of semiconducting polymer nanoparticles(Szymanski, C.; Wu, C.; Hooper, J.; Salazar, M. A.; Perdomo, A.; Dukes,A.; McNeill, J. D. J. Phys. Chem. B 2005, 109, 8543-8546; and Wu, C.;Szymanski, C.; McNeill, J. Langmuir 2006, 22, 2956-2960, the disclosuresof which are herein incorporated by reference in their entireties forall purposes), there has been rapid progress in the field, including thecharacterization of their complex photophysics (see, for example,Palacios, R. E.; Fan, F. R. F.; Grey, J. K.; Suk, J.; Bard, A. J.;Barbara, P. F. Nat. Mater. 2007, 6, 680-685; Wu, C.; Zheng, Y.;Szymanski, C.; McNeill, J. J. Phys. Chem. C 2008, 112, 1772-1781; Wu,C.; McNeill, J. Langmuir 2008, 24, 5855-5861; and Collini, E.; Scholes,G. D. Science 2009, 323, 369-373, the disclosures of which are hereinincorporated by reference in their entireties for all purposes), andtheir development for biological imaging and high resolutionsingle-particle tracking (see, for example, Wu, C. et al., J. J. Am.Chem. Soc. 2007, 129, 12904-12905; Wu, C. et al., J. ACS Nano 2008, 2,2415-2423; Wu, C.; et at, Chem. Int. Ed. 2009, 48, 2741-2745; Moon, J.H. et at, Chem. Int. Ed. 2007, 46, 8223-8225; Baler, M. C. et al, Am.Chem. Soc. 2009, 131, 14267-14273; Abbel, R.; et at, J. Chem. Commun.2009, 1697-1699; Pu, K. Y. et al, Chem. Mater. 2009, 21, 3816-3822; Yu,J. et al, J. Am. Chem. Soc. 2009, 131, 18410-18414; Howes, P. et al, J.Am. Chem. Soc. 2010, 132, 3989-3996; and Kim, S. et al. Chem. Commun.46, 1617-1619 (2010), the disclosures of which are herein incorporatedby reference in their entireties for all purposes).

Aspects of the present invention relate to a novel class offunctionalized fluorescent probes, referred to as functionalizedchromophoric polymer dots (functionalized CPdots or Pdots), and theirbiomolecular conjugates for a variety of applications, including but notlimited to flow cytometry, fluorescence activated sorting,immunofluorescence, immunohistochemistry, fluorescence multiplexing,single molecule imaging, single particle tracking, protein folding,protein rotational dynamics, DNA and gene analysis, protein analysis,metabolite analysis, lipid analysis, FRET based sensors, high throughoutscreening, cellular imaging, in vivo imaging, fluorescence-basedbiological assays such as immunoassays and enzyme-based assays, and avariety of fluorescence techniques in biological assays andmeasurements.

The unique properties of the functionalized chromophoric polymer dot hasbasis in, but is not limited to, high per-particle fluorescencebrightness, large absorption cross section, high fluorescence quantumyield, fast emissive rate, highly polarized fluorescence, excellentphotostability, and ease of storage. Upon conjugating with appropriatebiomolecules, the probe can be used in many areas, including but notlimited to flow cytometry, fluorescence activated sorting,immunofluorescence, immunohistochemistry, fluorescence multiplexing,single molecule imaging, single particle tracking, protein folding,protein rotational dynamics, DNA and gene analysis, protein analysis,metabolite analysis, lipid analysis, FRET based sensors, high throughoutscreening, cellular imaging, in vivo imaging, fluorescence-basedbiological assays such as immunoassays and enzyme-based assays, and avariety of fluorescence techniques in biological assays andmeasurements.

Additional advantages and features of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or can be learned by practice of the invention. Weperform experiments to demonstrate the present invention as illustratedin the following description and examples with reference to theaccompanying figures.

Pdots exhibit extraordinarily high fluorescence brightness under bothone-photon and two-photon excitations. Their brightness stems from anumber of favorable characteristics of semiconducting polymer molecules,including their large absorption cross-sections, fast emission rates,and high fluorescence quantum yields. Recent studies have also shownthat Pdots as fluorescent probes were photostable, and not cytotoxic indifferent cellular assays (see, for example, Wu, C.; Bull, B.;Szymanski, C.; Christensen, K.; McNeill, J. ACS Nano 2008, 2, 2415-2423;Pu, K. Y.; Li, K.; Shi, J. B.; Liu, B. Chem. Mater. 2009, 21, 3816-3822;and Rahim, N. A. A.; McDaniel, W.; Bardon, K.; Srinivasan, S.;Vickerman, V.; So, P. T. C.; Moon, J. H. Adv. Mater. 2009, 21,3492-3496, the disclosures of which are herein incorporated by referencein their entireties for all purposes).

However, for a wide range of biological applications, a significantproblem of Pdots has yet to be solved, namely how to control theirsurface chemistry and conjugation to biological molecules. Althoughresearch efforts involving silica or phospholipid encapsulation canresult in composite particles with surface functional groups (see, forexample, Wu, C.; Szymanski, C.; McNeill, J. Langmuir 2006, 22, 2956-2960and Howes, P.; Green, M.; Levitt, J.; Suhling, K.; Hughes, M. J. Am.Chem. Soc. 2010, 132, 3989-3996), all the reported results until now oncellular labeling with Pdots are presumably based on endocytosis (see,for example, Wu, C.; Bull, B.; Szymanski, C.; Christensen, K.; McNeill,J. ACS Nano 2008, 2, 2415-2423; Pu, K. Y.; Li, K.; Shi, J. B.; Liu, B.Chem. Mater. 2009, 21, 3816-3822; Howes, P.; Green, M.; Levitt, J.;Suhling, K.; Hughes, M. J. Am. Chem. Soc. 2010, 132, 3989-3996; andRahim, N. A. A.; McDaniel, W.; Bardon, K.; Srinivasan, S.; Vickerman,V.; So, P. T. C.; Moon, J. H. Adv. Mater. 2009, 21, 3492-3496), a farless effective and specific process compared to the established labelingmethods for organic fluorophores and Qdots. It is still unclear whetherPdot probes could be made specific enough to recognize cellular targetsfor effective labeling. This challenge thus far has severely preventedthe wide-spread use of Pdots in biological applications.

Advantageously, the present invention provides solutions for thechallenges associated with Pdot bioconjugation and specific cellulartargeting. In one aspect, the present invention provides a novelconjugation method that covalently links Pdots to biomolecules forlabeling cellular targets by specific antigen-antibody orbiotin-streptavidin interactions. This functionalization andbioconjugation strategy can be easily applied to any hydrophobic,fluorescent, semiconducting polymer. As shown in the examples providedherein, Pdot bioconjugates conjugated by the methods provided herein canbe used for single-particle imaging, cellular imaging, and flowcytometry experiments and their advantages over conventional organicfluorophores and Qdot probes are demonstrated. This work, therefore,opens up a new and practical pathway for employing a variety of highlyfluorescent, photostable, and non-toxic Pdot bioconjugates forbiological applications.

In one aspect, the present invention is based on a novel strategy forthe functionalization of Pdots, comprising entrapping heterogeneouspolymer chains into a single dot, driven by hydrophobic interactionsduring nanoparticle formation. A small amount of amphiphilic polymerbearing functional groups is co-condensed with the semiconductingpolymers to modify and functionalize the nanoparticle surface forsubsequent covalent conjugation to biomolecules, such as streptavidinand immunoglobulin G (IgG). The Pdot bioconjugates can effectively andspecifically label cellular targets, such as cell surface marker inhuman breast cancer cells, without any detectable non-specific binding.Single-particle imaging, cellular imaging, and flow cytometryexperiments indicate a much higher fluorescence brightness of Pdotscompared to those of Alex a dye and quantum dot probes. The successfulbioconjugation of these ultrabright nanoparticles presents a novelopportunity to apply versatile semiconducting polymers to variousfluorescence measurements in modern biology and biomedicine.

In one aspect, highly fluorescent semiconducting polymer dots withfunctional groups that allow for covalent conjugation to biomoleculesare provided. The strategy for functionalization of these Pdots is basedon entrapping heterogeneous polymer chains into a Pdot particle, drivenby hydrophobic interactions during nanoparticle formation. It is shownherein that a small amount of amphiphilic polymer bearing functionalgroups can be co-condensed with the semiconducting polymers to modifyand functionalize the nanoparticle surface. Subsequent covalentconjugation to biomolecules such as streptavidin and antibodies wereperformed using the standard carbodiimide coupling chemistry. These Pdotbioconjugates can effectively and specifically label cell-surfacereceptors and subcellular structures in both live and fixed cells,without any detectable non-specific binding. Single-particle imaging,cellular imaging, and flow cytometry were performed to experimentallyevaluate the Pdot performance, and demonstrate their high cellularlabeling brightness compared to those of Alexa-IgG and Qdot probes.These results bring forward a new class of highly fluorescentnanoparticle bioconjugates for a wide range of fluorescence-basedbiological detection.

Definitions

As used herein, the term “chromophoric nanoparticle” or “chromophoricpolymer dot” refers to a structure comprising one or more chromophoricpolymers that have been collapsed into a stable sub-micron sizedparticle. The chromophoric nanoparticles provided herein may be formedby any method known in the art for collapsing polymers, includingwithout limitation, methods relying on precipitation, methods relying onthe formation of emulsions (e.g. mini or micro emulsion), and methodsrelying on condensation. In a preferred embodiment, a chromophoricnanoparticle is formed by nanoprecipitation.

As used herein, “polymer” is a molecule composed of at least 2 repeatingstructural units typically connected by covalent chemical bonds.Polymers generally have extended molecular structures comprisingbackbones that optionally contain pendant side groups. It includeslinear polymer and branched polymer such as star polymers, combpolymers, brush polymers, ladders, and dendrimers.

As used herein, the term “chromophoric polymer” is a polymer in which atleast a portion of the polymer comprises chromophoric units. The term“chromophore” is given its ordinary meaning in the art. A chromophoreabsorbs certain wavelength of light from UV to near infrared region, andmay be or may not be emissive.

A “chromophoric unit” in this invention includes, but not limited to,unit of structures with delocalized pi-electrons, unit of small organicdye molecules, and unit of metal complexes. Accordingly, examples ofchromophoric polymers include polymers comprising units of structureswith delocalized pi-electrons such as semiconducting polymers, polymerscomprising units of small organic dye molecules, polymer comprisingunits of metal complexes, and polymers comprising unit of anycombinations thereof.

As used herein, the term “functional group” refers to any chemical unitthat can be attached, such as by any stable physical or chemicalassociation, to the chromophoric polymer, thereby rendering the surfaceof the chromophoric polymer dot available for conjugation. Non-limitingexamples of functional groups include, carboxylic acid, amino, mercapto,azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate,phosphate, cyanate, succinimidyl ester, alkyne, strained alkyne, azide,diene, alkene, cyclooctyne, and phosphine groups, substitutedderivatives thereof, and combinations there of.

As used herein, the term “functionalization agent” refers to anymolecule that can be attached, such as by any stable physical orchemical association, to the core of a chromophoric polymer dot,providing a functional group on the surface of the polymer dot.

As used herein the term “hydrophilic functional group” refers either toa functional group that is hydrophilic in nature or to a hydrophobicfunctional group that is attached to a hydrophilic side chain orhydrophilic moiety, which renders the hydrophobic functional group morehydrophilic in nature and which facilitate the arrangement of thehydrophobic functional groups on the chromophoric polymer dot particlesurface rather than getting buried inside the hydrophobic core of thechromophoric polymer dot. Examples of hydrophobic functional groups thatcan be rendered more hydrophilic by attachment to hydrophilic sidechains or moieties include but not limited to alkyne, strained alkyne,azide, diene, alkene, cyclooctyne, and phosphine groups (for clickchemistry) attached to a hydrophilic side chain such as PEG(polyethylene glycol) or to any other hydrophilic side chains.

As used herein, the term “bioorthogonal reaction” refers to aconjugation between non-native, non-perturbing chemical handles that canbe modified in living systems through highly selective reactions withexogenously delivered probes. The most well known of the bioorthogonalreaction schemes is known as click chemistry. For review ofbioorthogonal reaction schemes, see, for example Best M D, Biochemistry.2009 Jul. 21; 48(28):6571-84, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

As used herein, the term “click reaction” is recognized in the art,which describe a collection of supremely reliable and self-directedorganic reactions, such as the most recognized copper catalyzedazide-alkyne [3+2] cycloaddition. Non-limiting examples of clickchemistry reactions can be found, for example, in H. C. Kolb, M. G.Finn, K. B. Sharpless, Angew. Chem. Int. Ed. 2001, 40, 2004 and E. M.Sletten, C. R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974, thedisclosures of which are herein incorporated by reference in theirentireties for all purposes.

As used herein, the term “cross-linking agent” is used to describe acompound that is capable of forming a chemical bond between moleculargroups on similar or dissimilar molecules so as to covalently bondtogether the molecules. Examples of common cross-linking agents areknown in the art. See, for example, Bioconjugate Techniques (AcademicPress, New York, 1996 or later versions) the content of which is hereinincorporated by reference in its entirety for all purposes. Indirectattachment of the biomolecule to monovalent chromophoric polymer dotscan occur through the use of “linker” molecule, for example, avidin,streptavidin, neutravidin, biotin or a like molecule.

As used herein, an “antibody” refers to a polypeptide comprising aframework region from an immunoglobulin gene or fragments thereof thatspecifically binds and recognizes an antigen. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon, and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. Typically, the antigen-bindingregion of an antibody will be most critical in specificity and affinityof binding. Antibodies can be polyclonal or monoclonal, derived fromserum, a hybridoma or recombinantly cloned, and can also be chimeric,primatized, or humanized.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H) ⁻C_(H) ¹ by adisulfide bond. The F(ab)′₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region, thereby converting theF(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially Fabwith part of the hinge region (see Fundamental Immunology (Paul ed., 3ded. 1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature, 348:552-554(1990)).

Functionalized Chromophoric Polymer Dots

The present invention provides, in one aspect, a functionalizedchromophoric polymer dot, which comprises a core and a cap. The “core”contains at least one chromophoric polymer. The “cap” comprises afunctionalization agent. The functionalization agent is attached to thecore of chromophoric polymer by physical association or chemicalbonding, and provides surface functional groups on chromophoric polymerdot for bioconjugation. In another embodiment, the functionalizedchromophoric polymer dot comprises just the core attached covalently tofunctional groups which facilitate bioconjugation.

In one aspect, the present invention provides a functionalizedchromophoric polymer dot (Pdot) comprising a core and a cap, whereinsaid core comprises a chromophoric polymer, and said cap comprises afunctionalization agent bearing one or more functional groups, with theproviso that not all of the said cap is an organo silicate. In aspecific embodiment, the cap does not comprise an organo silicate.

In one embodiment, the present invention provides a functionalizedchromophoric polymer dot that comprises a core of chromophoric polymerranging in size from about 1 nm to about 1000 nm, and an amphiphilicfunctionalization layer, wherein the hydrophobic moiety is permanentlyanchored to the core of polymer dot through hydrophobic interaction andthe hydrophilic functional groups such as carboxylic acid extend insolution for further bioconjugation.

In a specific embodiment, the present invention provides afunctionalized chromophoric polymer dot (Pdot) having a hydrophobic coreand a hydrophilic cap, the Pdot comprising: (a) a chromophoric polymer;and (b) an amphiphilic molecule, having a hydrophobic moiety and ahydrophilic moiety attached to a reactive functional group, wherein thechromophoric polymer is embedded within the hydrophobic core of the Pdotand; wherein a portion of the amphiphilic molecule is embedded withinthe core of the Pdot and the reactive functional group is located in thehydrophilic cap. In a preferred embodiment, the chromophoric polymer isa semiconducting polymer.

In one embodiment of the chromophoric polymer dots provided herein, thereactive functional group is selected from the group consisting of acarboxyl, amino, mercapto, azido, alkyne, aldehyde, hydroxyl, carbonyl,sulfate, sulfonate, phosphate, cyanate, succinimidyl ester, and aderivative thereof. In a specific embodiment, the reactive functionalgroup is an alkyne containing moiety, azido containing moiety, or othermoiety capable of being conjugated to a molecule via a click chemistryreaction.

In one embodiment, the chromophoric polymer is a semiconductinghomopolymer. Many semiconducting homopolymers are known in the art,including without limitation, fluorene polymers, phenylene vinylenepolymers, phenylene polymers, phenylene ethynylene polymers,benzothiazole polymers, thiophen polymers, carbazole fluorene polymers,boron-dipyrromethene-based polymers, and derivatives thereof. A list ofcommon semiconducting polymers and their abbreviations is given in Table1.

TABLE 1 Non-limiting examples of semiconducting polymers FluorenePolymers: Poly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF),Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO), Fluorene based Copolymers:Poly[{9,9-diocty1-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethyl hex yloxy)-1,4- phenylene}] (PFPV),Poly[(9,9-dioctylfluoreny1-2,7-diy1)-co-(1,4-benzo-{2,1,3}-thiadiazole)](PFBT),Poly[(9,9-dioctylfluoreny1-2,7-diy1)-co-(4,7-Di-2-thieny1-2,1,3-benzothiadiazole){(PFTBT),Poly[(9,9-dioctylfluoreny1-2,7-diy1)_(0.9)-co-(4,7-Di-2-thieny1-2,1,3-benzothiadiazole)_(0.1)](PF- 0.1TBT), Phenylene Vinylene Polymers:Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV),Phenylene Ethynylene PolynmersPoly(2,5-di(3′,7′-dimethyloctyl)phenylene-1,4-ethynylene (PPE),

In another embodiment, the chromophoric polymer is a semiconductingcopolymer comprising at least two different chromophoric units. Forexample, a chromophoric copolymer may contain both fluorene andbenzothiazole chromophoric units present at a given ratio. Typicalchromophoric units used to synthesize semiconducting copolymers include,but are not limited to fluorene unit, phenylene vinylene unit, phenyleneunit, phenylene ethynylene unit, benzothiazole unit, thiophen unit,carbazole fluorene unit, boron-dipyrromethene unit, and derivativesthereof. The different chromophoric units may be segregated, as in ablock copolymer, or intermingled. As used herein, a chromophoriccopolymer is represented by writing the identity of the majorchromophoric species. For example, PFBT is a chromophoric polymercontaining fluorene and benzothiazole units at a certain ratio. In somecases, a dash is used to indicate the percentage of the minorchromophoric species and then the identity of the minor chromophoricspecies. For example, PF-0.1BT is a chromophoric copolymer containing90% PF and 10% BT.

In other embodiments, a chromophoric polymer dot comprises a blend ofsemiconducting polymers. The blends may include any combination ofchromophoric homopolymers, copolymers, and oligomers. Chromophoricpolymer blends used to form polymer dots may be selected in order totune the properties of the resulting polymer dots, for example, toachieve a desired excitation or emission spectra for the polymer dot.

In one embodiment of the chromophoric polymer dots provided herein, thefunctionalization agent is an amphiphilic molecule. In certainembodiment, the amphiphilic molecule is a chromophoric polymer that hasbeen modified with a functional group. For example, in one embodiment,the present invention provides a functionalized chromophoric polymer dot(Pdot) having a hydrophobic core and a hydrophilic cap, the Pdotcomprising: (a) a first semiconducting polymer; and (b) a secondsemiconducting polymer attached to a reactive functional group, whereinthe first semiconducting polymer is embedded within the hydrophobic coreof the Pdot and; wherein a portion of the second semiconducting polymeris embedded within the core of the Pdot and the reactive functionalgroup is located in the hydrophilic cap (i.e., on the surface of thePdot).

In another embodiments, the amphiphilic molecule is a non-chromophoricmolecule, for example a non-chromophoric polymer, lipid, carbohydrate,or other non-chromophoric molecule modified with a reactive functionalgroup. For example, in one embodiment, the present invention provides afunctionalized chromophoric polymer dot (Pdot) having a hydrophobic coreand a hydrophilic cap, the Pdot comprising: (a) a semiconductingpolymer; and (b) an amphiphilic molecule, having a hydrophobic moietyand a hydrophilic moiety attached to a reactive functional group,wherein the semiconducting polymer is embedded within the hydrophobiccore of the Pdot and; wherein a portion of the amphiphilic molecule isembedded within the core of the Pdot and the reactive functional groupis located in the hydrophilic cap (i.e., on the surface of the Pdot).

In a specific embodiment, the amphiphilic polymer is an amphiphiliccomb-like polymer, for example, a polystyrene based comb-likeamphiphilic polymer or a poly(methyl methacrylate) based comb-likepolymers. In another specific embodiment, the amphiphilic polymer ispoly(styrnene-co-maleic anhydride) (PSMA).

In a specific embodiment, the amphiphilic polymer is a polystyrene basedcomb-like polymers. Non limiting examples of polystyrene based comb-likepolymers include, polystyrene graft acrylic acid, polystyrene graftethylene oxide functionalized with carboxy, polystyrene graft ethyleneoxide functionalized with amine, polystyrene graft ethylene oxidefunctionalized with thiol, polystyrene graft ethylene oxidefunctionalized with succinimidyl ester, polystyrene graft ethylene oxidefunctionalized with azide, polystyrene graft ethylene oxidefunctionalized with alkyne, polystyrene graft ethylene oxidefunctionalized with cyclooctyne, polystyrene graft ethylene oxidefunctionalized with ester, phosphine, polystyrene graft butyl alcohol,and the like. In a specific embodiment, the amphiphilic polymer is apolyethylene glycol-grafted polystyrene. In a more specific embodiment,the polyethylene glycol moiety of the amphiphilic polymer is attached toone or more carboxyl groups. In another specific embodiment, theamphiphilic polymer is a poly(styrene-co-maleic anhydride).

In another embodiment, the amphiphilic polymer is a poly(methylmethacrylate) based comb-like polymers. Non-limiting examples ofpoly(methyl methacrylate) based comb-like polymers include, poly(methylmethacrylate) graft acrylic acid, poly(methyl methacrylate) graftethylene oxide functionalized with carboxy, poly(methyl methacrylate)graft ethylene oxide functionalized with amine, poly(methylmethacrylate) graft ethylene oxide functionalized with thiol,poly(methyl methacrylate) graft ethylene oxide functionalized withsuccinimidyl ester, poly(methyl methacrylate) graft ethylene oxidefunctionalized with azide, poly(methyl methacrylate) graft ethyleneoxide functionalized with alkyne, poly(methyl methacrylate) graftethylene oxide functionalized with cyclooctyne, poly(methylmethacrylate) graft ethylene oxide functionalized with ester,poly(methyl methacrylate) graft ethylene oxide functionalized withphosphine, and the like.

In yet another embodiment, the amphiphilic polymer is a comb-likepolymer comprising a carboxyl, amine, thiol, ester, succinimidyl ester,azide, alkyne, cyclooctyne, and/or phosphine group.

In some embodiments, the amphiphilic polymer is an amphiphiliccopolymer, for example, (1) poly((meth)acrylic acid) based copolymerssuch as: poly(acrylic acid-b-acrylamide), poly(acrylic acid-b-methylmethacrylate), poly(acrylic acid-b-N-isopropylacrylamide),poly(n-butylacrylate-b-acrylic acid), pol y(sodium acrylate-b-methylmethacrylate), poly(methacrylic acid-b-neopentyl methacrylate),poly(methyl methacrylate-b-acrylic acid), poly(methylmethacrylate-b-methacrylic acid), poly(methylmethacrylate-b-N,N-dimethyl acrylamide), poly(methylmethacrylate-b-sodium acrylate), poly(methyl methacrylate-b-sodiummethacrylate), poly(neopentyl methacrylate-b-methacrylic acid),poly(t-butyl methacrylate-b-ethylene oxide),poly(2-acrylamido-2-methylpropanesulfonic acid-b-acrylic acid); (2)polydiene based copolymers such as: poly(butadiene(1,2addition)-b-ethylene oxide), poly(butadiene(1,2addition)-b-methylacrylic acid, poly(butadiene(1,4 addition)-b-acrylicacid), poly(butadiene(1,4 addition)-b-ethylene oxide, poly(butadiene(1,4addition)-b-sodium acrylate), poly(butadiene(1,4 addition)-b-N-methyl4-vinyl pyridinium iodide), poly(isoprene-b-ethylene oxide),poly(isoprene-b-ethylene oxide), and poly(isoprene-b-N-methyl 2-vinylpyridinium iodide); (3) poly(ethylene oxide) based copolymers such as:poly(ethylene oxide-b-acrylic acid), poly(ethylene oxide-b-acrylamide),poly(ethylene oxide-b-butylene oxide), poly(ethyleneoxide-b-ε-caprolactone), poly(ethylene oxide-b-lactide), poly(ethyleneoxide-b-lactide), poly(ethylene oxide-b-methacrylic acid), poly(ethyleneoxide-b-methyl acrylate), poly(ethylene oxide-b-N-isopropylacrylamide),poly(ethylene oxide-b-methyl methacrylate), poly(ethyleneoxide-b-nitrobenzyl methacrylate), poly(ethyleneoxide-b-N,N-dimethylaminoethylmethacrylate), poly(ethyleneoxide-b-propylene oxide), poly(ethylene oxide-b-t-butyl acrylate),poly(ethylene oxide-b-t-butyl methacrylate), poly(ethyleneoxide-b-tetrahydrofurfuryl methacrylate), poly(ethylene oxide-b-2-ethyloxazoline), poly(ethylene oxide-b-2-hydroxyethyl methacrylate),poly(ethylene oxide-b-2-methyl oxazoline); (4) polyisobutylene basedcopolymers such as poly(isobutylene-b-acrylic acid),poly(isobutylene-b-ethylene oxide), poly(isobutylene-b-methacrylicacid); (5) polystyrene based copolymers such aspoly(styrene-b-acrylamide), poly(styrene-b-acrylic acid),poly(styrene-b-cesium acrylate), poly(styrene-b-ethylene oxide),poly(styrene-b-ethylene oxide) acid cleavable at the block junction,poly(styrene-b-methacrylic acid), poly(4-styrenesulfonic acid-b-ethyleneoxide), poly(styrenesulfonic acid-b-methylbutylene),poly(styrene-b-N,N-dimethylacrylamide), poly(styrene-b-N-isopropylacrylamide), poly(styrene-b-N-methyl 2-vinyl pyridinium iodide),poly(styrene-b-N-methyl-4-vinyl pyridinium iodide),poly(styrene-b-propylacrylic acid), poly(styrene-b-sodium acrylate)poly(styrene-b-sodium methacrylate), poly(p-chloromethylstyrene-b-acrylamide), poly(styrene-co-p-chloromethylstyrene-b-acrylamide), poly(styrene-co-p-chloromethyl styrene-b-acrylicacid), poly(styrene-b-methylbutylene-co-isoprene sulfonate); (6)polysiloxane based copolymers such as poly(dimethylsiloxane-b-acrylicacid), poly(dimethylsiloxane-b-ethylene oxide),poly(dimethylsiloxane-b-methacrylic acid); (7)poly(ferrocenyldimethylsilane) based copolymers such as poly(ferrocenyldimethylsilane-b-ethylene oxide); (8) poly(2-vinylnaphthalene) based copolymers such as poly(2-vinyl naphthalene-b-acrylicacid), (9) poly (vinyl pyridine and N-methyl vinyl pyridinium iodide)based copolymers such as poly(2-vinyl pyridine-b-ethylene oxide),poly(2-vinyl pyridine-b-methyl acrylic acid), poly(N-methyl 2-vinylpyridinium iodide-b-ethylene oxide), poly(N-methyl 4-vinyl pyridiniumiodide-b-methyl methacrylate), poly(4-vinyl pyridine-b-ethylene oxide)PEO end functional OH; (10) poly(vinyl pyrrolidone) based copolymerssuch as poly(vinyl pyrrolidone-b-D/L-lactide); and the like.

In one embodiment, the hydrophilic moiety of an amphiphilic polymer usedto functionalize a chromophoric polymer dot is a water soluble polymer,such as a polyalkylene glycol (e.g., a PEG), a PEO, a polypropyleneglycol, a polyoxyalkylene, a starch, a poly-carbohydrate, a polysialicacid, and the like. In one embodiment, the water soluble polymer is apolyalkylene glycol. In a more specific embodiment, the water solublemoiety is a polyethylene glycol (a PEG).

In one embodiment, the hydrophobic moiety of the amphiphilic polymerused to functionalize a chromophoric polymer dot is a hydrophobicpolymer. Non-limiting examples of polymers that may be used include,without limitation, poly(meth)acrylate polymers, polyacrylamidepolymers, polyisobutylene, polydiene, polyphenylene, polyethylene,poly(ethylene glycol), polylactide, polystyrene, polysiloxane,poly(vinyl pyridine), poly(vinylpyrrolidone), polyurethane, a blockcopolymer thereof, a random or alternating copolymer thereof, and thelike.

Dependent upon many factors, such as, the desired level offunctionalization, desired spectrophotometric properties, and intendeduse, the weight ratio of amphiphilic molecule to chromophoric polymer ina functionalized chromophoric polymer dot as provided herein, will rangefrom about 0.01% to about 50%. In a preferred embodiment, the weightratio of amphiphilic molecule to chromophoric polymer is between about5% and about 20%. In yet other embodiment, the weight ratio ofamphiphilic molecule to chromophoric polymer is no larger than about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, or about 50%. In a specific embodiment, the weightratio of amphiphilic molecule to chromophoric polymer is no larger thanabout 25%.

In one embodiment, the chromophoric polymer dots provided herein furthercomprise a component selected from the group consisting of a fluorescentdye, an inorganic luminescent material, a magnetic material, and a metalembedded within the core of the polymer dot.

In yet other embodiments, a chromophoric polymer dot, as providedherein, may be further conjugated to one or more non-reactive chemicalgroups. In this fashion, the presence of non-reactive chemical groups onthe surface of the polymer dot will reduce and/or eliminate non-specificassociations between the surface of the polymer dots and othermolecules, e.g., proteins. In one embodiment, the non-reactive chemicalgroup is a water soluble polymer, such as a polyalkylene glycol (e.g., aPEG), a PEO, a polypropylene glycol, a polyoxyalkylene, a starch, apoly-carbohydrate, a polysialic acid, and the like. In one embodiment,the water soluble polymer is a polyalkylene glycol. In a more specificembodiment, the water soluble moiety is a polyethylene glycol (a PEG).

Utilizing the functionalization scheme described herein, successfulconjugations of streptavidin and/or antibodies to different types ofPdots, including the five Pdots described in Wu et al. (Wu, C.; Bull,B.; Szymanski, C.; Christensen, K.; McNeill, J. ACS Nano 2008, 2,2415-2423), have been achieved.

The present invention provides, among other aspects and embodiments,functionalized PFBT dots for single-particle imaging, cellular labeling,and flow cytometry applications. Functionalized PFBT dots exhibit arelatively broad absorption peak around 460 nm (FIG. 3), which is aconvenient wavelength region for fluorescence microscopy and laserexcitations. Analysis of the absorption and fluorescence spectra from˜10 nm-diameter PFBT dots indicated a peak extinction coefficient of1.5×10⁷ M⁻¹ cm⁻¹ and a fluorescence quantum yield of 0.30. Thephotophysical properties of PFBT dots are summarized in Table 2,together with the properties of two widely used probes purchased fromInvitrogen: Qdot 565 and fluorescent IgG-Alexa 488 (˜6 dye molecules perIgG). These two commercial probes were selected because they haveemissions in a similar wavelength region as that of PFBT dots. It isimportant to note that Pdots contain multiple emitters, thus, althoughthe lifetimes of Pdots are ˜50 times shorter than Qdots, the emissionrates of Pdots can be three orders of magnitude faster than Qdots (see,Id.).

TABLE 2 Photophysical Properties of PFBT dots, IgG-Alexa 488, and Qdot565. Probe PFBT dot Alexa 488 Qdot 565 (Size) (~10 nm) (~1 nm) (~15 nm)Abs./Fluores. Max 460 nm/540 nm 496 nm/519 nm UV/565 nm ExtinctionCoeffocient 1.0 × 10⁷ M⁻¹ cm⁻¹ 5.3 × 10⁴ M⁻¹ cm⁻¹ 2.9 × 10⁵ M⁻¹ cm⁻¹ at488 nm Quantum Yield 0.3 0.9 0.3~0.5 Fluorescence Lifetime 0.6 ns 4.2 ns~20 ns

The data for Alexa 488 and Qdot 565 given in Table 2 are according tothe probe specification provided by Invitrogen. The parameters of Alexa488 are for single-dye molecules. An IgG-Alexa 488 probe has ahydrodynamic diameter of 12 nm, contains an average of 6 dye molecules,but its brightness corresponds to ˜2-4 dye molecules due toself-quenching. Fluorescence lifetime of PFBT dots was measured by aTCSPC setup. Also note that single PFBT dots contain multiple emitters,which results in photon emission rates that are higher than thosepredicted from fluorescence lifetime alone.

The “Core”

In one embodiment, the core of the chromophoric polymer dot comprises aluminescent semiconducting polymer with delocalized pi-electrons. Theterm “semiconducting polymer” is recognized in the art. Typicalluminescent semiconducting polymers include, but are not limited tofluorene polymers, phenylene vinylene polymers, phenylene polymers,phenylene ethynylene polymers, benzothiazole polymers, thiophenpolymers, carbazole fluorene polymers, boron-dipyrromethene-basedpolymers, polymer derivatives thereof, and copolymers comprising anycombinations thereof. In some embodiments, the chromophoric polymers maybe semiconducting polymers covalently linked with unit of small organicdyes, or metal complexes as emissive species. Such emissive units cantune the emission color, increase the quantum yield, and improve thephotostability of the chromophoric polymer dot.

In a preferred embodiment, the small organic dyes, or metal complexescan have sensing functions, and therefore add additional functionalitiesto the chromophoric polymer dot, such as oxygen sensing capability, ionsensing capability, glucose sensing capability, neurotransmitter sensingcapability, drug sensing capability, metabolite sensing capability,protein sensing capability, signaling molecule sensing capability, toxinsensing capability, DNA and RNA sensing capability, and the like. Forexample, platinum porphyrin can be linked covalently to semiconductingpolymers, and the resulting chromophoric polymer dots can be used as anoxygen sensor. In another embodiment, the chromophoric polymers may besemiconducting polymers covalently linked with unit of photochromic dye.A photochromic unit can serve as energy transfer acceptor. Upon lightillumination of appropriate wavelengths, the photochromic unit undergoesa reversible transformation between two structural forms with differentabsorption spectra. Accordingly, the luminescence of chromophoricpolymer dot can be modulated with another wavelength of light, makingthe chromophoric polymer dot have a desirable photoswitching capability.The photoswitching probes are particularly useful for super-resolutionimaging techniques such as PALM, STORM, etc as recognized in the art.

In some embodiments, the core of the chromophoric polymer dot containspolymers comprising unit of small organic dye molecules, metalcomplexes, photochromic dye, and any combinations thereof, for example,optically inactive polymer such as polystyrene covalently linked orgrafted with small organic dye, metal complexes, photochromic dyes andany combination thereof. These dyes or metal complexes may have sensingfunctions, and therefore add additional functionalities to thechromophoric polymer dot, such as oxygen sensing capability, ion sensingcapability, glucose sensing capability, neurotransmitter sensingcapability, drug sensing capability, metabolite sensing capability,protein sensing capability, signaling molecule sensing capability, toxinsensing capability, DNA and RNA sensing capability, and the like.

In a related embodiment, a chromophoric polymer dot as provided hereinmay include one or more pH sensitive dyes. For example, fluorescein is awell known pH sensitive dye that may be conjugated to the surface of apolymer dot provided herein. Once fluorescein is conjugated to thepolymer dot, it can participate in fluorescence resonance energytransfer (FRET) with the chromophoric polymers present in the core ofthe polymer dot. When present in solution, the FRET emission of afluorescein-conjugated chromophoric polymer dot will be dependent uponthe pH of the solution. This is demonstrated in Example 31 providedherein.

Likewise, in one embodiment, the present invention provides a method fordetermining the pH of a solution, the method comprising the steps ofcontacting the solution with a chromophoric polymer dot conjugated to apH sensitive dye, or having a pH sensitive dye embedded within the coreof the polymer dot, capable of participating in FRET with chromophoricunits present in the core of the polymer dot, exciting chromophoricunits in the core of the polymer dot, and detecting the FRET emission ofthe pH sensitive dye.

In another related embodiment, a chromophoric polymer dot as providedherein may include one or more temperature sensitive dyes. For example,Rhodamine B is a well known temperature sensitive dye that may beembedded within the core or functionalized to the surface of achromophoric polymer dot. Once embedded within the hydrophobic core ofthe polymer dot, it can participate in fluorescence resonance energytransfer (FRET) with the chromophoric polymers present in the core ofthe polymer dot. When present in solution, the FRET emission of aRhodamine B-conjugated chromophoric polymer dot will be dependent uponthe temperature of the solution. This is demonstrated in Example 30provided herein.

Likewise, in one embodiment, the present invention provides a method fordetermining the temperature of a solution, the method comprising thesteps of contacting the solution with a chromophoric polymer dotconjugated to a temperature sensitive dye, or having a temperaturesensitive dye embedded within the core of the polymer dot, capable ofparticipating in FRET with chromophoric units present in the core of thepolymer dot, exciting chromophoric units in the core of the polymer dot,and detecting the FRET emission of the temperature sensitive dye.

The core may be solely semiconducting homopolymer, semiconductingcopolymer, or semiconducting oligomer composed of at least 2 repeatingstructural units. The core may also comprise simultaneously two or moreof semiconducting homopolymer, semiconducting copolymer, and/orsemiconducting oligomer. The semiconducting polymer or oligomerpreferably has light-emitting properties. Typical light-emittingpolymers include, but are not limited to fluorene polymers, phenylenevinylene polymers, phenylene polymers, phenylene ethynylene polymers,benzothiazole polymers, thiophen polymers, carbazole fluorene polymers,boron-dipyrromethene-based polymers, polymer derivatives thereof, andcopolymers comprising any combinations thereof.

The core may also comprise a semiconducting polymer as activefluorophore, physically mixed or chemically cross-linked with otheroptically inactive polymers, for example polystyrene, to have desirableproperties such as polarized emission. The optically inactive polymermay contain functional groups for conjugation to the desiredbiomolecule, and thus render the chromophoric polymer dots the abilityto associate with a biomolecule of interest.

The core may also comprise semiconducting polymer, physically mixed orchemically cross-linked with other chromophoric polymer such asoptically inactive polymer covalently linked or grafted with smallorganic dye, metal complexes, photochromic dyes and any combinationthereof, to have additional functionalities such as oxygen sensingcapability, ion sensing capability, glucose sensing capability,neurotransmitter sensing capability, drug sensing capability, metabolitesensing capability, protein sensing capability, signaling moleculesensing capability, toxin sensing capability, DNA and RNA sensingcapability, and the like. The core may also comprise a semiconductingpolymer, physically mixed or chemically cross-linked with othercomponents consisting of fluorescent dye, inorganic luminescentmaterials, magnetic materials, metal materials, to tune emission color,improve quantum yield and photostability, and have additionalfunctionalities such as magnetic functions, plasmon resonance functions,and the like. The size of the core of a chromophoric polymer dot rangesand can be tuned in size from about 1 nm to about 1000 nm. FIG. 1 showsa chemical structure of chromophoric copolymer PFBT derived fromfluorene and benzothiazole.

The “Cap”

The cap of a chromophoric polymer dot comprises a functionalizationagent attached to the core of chromophoric polymer by physicalassociation or chemical bonding, and provides surface functional groupson chromophoric polymer dot for bioconjugation. Preferably, thefunctionalization agent is a polymer, which may or may not bechromophoric. For example, functionalization can be provided by anamphiphilic polymer that comprises a hydrophobic moiety and ahydrophilic moiety containing one or more functional groups. In oneembodiment, the hydrophobic moiety is physically embedded in the core ofthe chromophoric polymer dot by hydrophobic interaction, while thehydrophilic moiety containing functional groups extends into thesolution for bioconjugation. In another embodiment, it may be preferredto chemically associate the polymer containing the functional groupswith a preformed chromophoric polymer to form the functionalizedchromophoric polymer dot. Chemical association can be any number ofchemical bonding interactions, such as a covalent bond, an ionic bond, apolar covalent bond, a hydrogen bond, or metal-ligand bond.

Preferably, the functionalization agent will help maintain the watersolubility and stability of the chromophoric polymer dot without causingaggregation for at least about one week, and more preferably stable insolution for over 1 month, 3 months, 6 months, 1 year, 3 years, and 5years.

In one embodiment, the functionalization agent is attached to the coreof chromophoric polymer by chemical association such as covalent bond,ionic bond, hydrogen bond and the like. Functionalization agent may bechemically linked to reactive sites in the backbone or side chain of thechromophoric polymer.

In another embodiment, the functionalization agent is anchored to thecore of chromophoric polymer dot by physical association. Physicalassociation can arise from a range of forces, including but not limitedto van de Waals, electrostatic, pi-stacking, hydrophobic, entropicforces and combinations thereof. In a preferable embodiment, thefunctionalization agent may comprise a hydrophobic moiety and ahydrophilic moiety containing one or more functional groups. Thehydrophobic moiety is physically embedded in the chromophoric polymerdots by hydrophobic interaction, while the hydrophilic moiety containingfunctional groups extends into the solution for bioconjugation. In aparticular embodiment, the functionalization agent can be any moleculethat comprises biotin, folic acid, folate, phalloidin, or a peptide, anucleic acid, a carbohydrate, and the like, which can directly orindirectly bind to biological entities.

In some embodiments, the functionalization agent is a small organicmolecule, a surfactant, or a lipid molecules that comprise functionalgroups such as carboxylic acid or salts thereof, amino, mercapto, azido,alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate, phosphate,cyanate, succinimidyl ester, substituted derivatives thereof, orcombinations there of. In general, any functional groups that allowbioconjugation may be used. Such groups could be found by one ofordinary skill in the art, for example in Bioconjugate Techniques(Academic Press, New York, 1996 or later versions). These molecules canbe attached chemically or physically to the core of chromophoric polymerdot, and provide surface functional groups on the chromophoric polymerdot for bioconjugation.

In another embodiment, the functionalization agent is a polymer thatcomprises functional groups such as carboxylic acid or salts thereof,amino, mercapto, azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate,sulfonate, phosphate, cyanate, succinimidyl ester substitutedderivatives thereof, or combinations there of. In general, anyfunctional groups that allow bioconjugation may be used. Such groupscould be found by one of ordinary skill in the art, for example inBioconjugate Techniques (Academic Press, New York, 1996 or laterversions). The functionalization polymer is associated with the core ofchromophoric polymer dot by any chemical bonding or physical forces, andprovide surface functional groups on the chromophoric polymer dot forbioconjugation.

In a preferred embodiment, the functionalization agent is an amphiphilicpolymer that comprises a hydrophobic moiety and a hydrophilic moietycontaining one or more functional groups. The hydrophobic moiety isphysically embedded in the chromophoric polymer dots by hydrophobicinteraction, while the hydrophilic moiety containing functional groupsextends into the solution for bioconjugation.

In certain embodiment, the amphiphilic polymer is a hydrophobicchromophoric polymer that has been modified with hydrophilic functionalgroups. For example, in one embodiment, the functionalized chromophoricpolymer dot (Pdot) having a hydrophobic core and a hydrophilic cap, thePdot comprising: (a) a first hydrophobic semiconducting polymer; and (b)a second amphiphlic semiconducting polymer attached to a reactivefunctional group, wherein the first hydrophobic semiconducting polymeris embedded within the hydrophobic core of the Pdot and; wherein aportion of the second semiconducting polymer is embedded within the coreof the Pdot and the reactive functional group is located in thehydrophilic cap (i.e., on the surface of the Pdot).

In another embodiment, the functionalization agent is a comb-likeamphiphilic polymer comprising multiple repeating units of hydrophobicmoiety and multiple repeating units of hydrophilic moiety so that thefunctionalization agent is permanently anchored to the core of polymerdot by strong hydrophobic force and the hydrophilic moieties comprisefunctional groups that extend into the solution for bioconjugation.

The hydrophobic moiety of an amphiphilic polymer may be an alkyl group,more preferably an aryl group to strengthen the hydrophobic attachmentto the core of polymer dot that consist of many aromatic rings. Suitablehydrophilic moieties may be polyalkylene glycol which may befunctionalized by carboxylic acid or salts thereof, amino, mercapto,azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate,phosphate, cyanate, succinimidyl ester, substituted derivatives thereof,or combinations there of. In general, any functional groups that allowbioconjugation may be used. Such groups could be found by one ofordinary skill in the art, for example in Bioconjugate Techniques(Academic Press, New York, 1996 or later versions). Desirably, thefunctionalization agent of the present invention is an amphiphilicpolymer that comprises multiple units of an aryl group and multipleunits of carboxylic acid, amino, mercapto, azido, alkyne, aldehyde,succinimidyl ester, or hydroxyl attached to the polyalkylene glycol.FIG. 1 shows the chemical structure of such a functionalization agent,polystyrene graft ethylene oxide terminated with carboxylic acid(PS-PEG-COOH).

Bioorthogonal or Clickable Chromophoric Polymer Dots

Click chemistry describes a powerful set of chemical reactions that arerapid, selective, and produce high yields (H. C. Kolb, M. G. Finn, K. B.Sharpless, Angew. Chem. Int. Ed. 2001, 40, 2004). The most recognized ofthese reactions is the copper (I)-catalyzed azide-alkyne cycloaddition,which has been applied to diverse areas, ranging from materials science(J. E. Moses, A. D. Moorhouse, Chem. Soc. Rev. 2007, 36, 1249) to drugdiscovery (H. C. Kolb, K. B. Sharpless, Drug Discov. Today 2003, 8,1128) and chemical biology (Q. Wang, T. R. Chan, R. Hilgraf, V. V.Fokin, K. B. Sharpless, M. G. Finn, J. Am. Chem. Soc. 2003, 125, 3192;A. E. Speers, G. C. Adam, B. F. Cravatt, J. Am. Chem. Soc. 2003, 125,4686; J. A. Prescher, C. R. Bertozzi, Nat. Chem. Biol. 2005, 1, 13; N.J. Agard, J. M. Baskin, J. A. Prescher, A. Lo, C. R. Bertozzi, ACS Chem.Biol. 2006, 1, 644; and E. M. Sletten, C. R. Bertozzi, Angew. Chem. Int.Ed. 2009, 48, 6974).

For biological applications, both azido and alkyne groups are consideredto be bioorthogonal chemical reporters because they do not interact withany native biological functional groups. As a result, thesebioorthogonal reporters can be incorporated into a target biomoleculeusing the cell's biosynthetic machinery to provide chemical handles thatcan be subsequently tagged with exogenous probes. The bioorthogonalchemical reporters are complementary to genetically encoded tags, suchas green fluorescent protein (GFP), and provide a means to tagbiomolecules without the need of direct genetic encoding. Thesereporters offer a powerful approach for visualizing multiple classes ofbiomolecules, not just proteins but also glycans, lipids, and nucleicacids (Prescher et al., supra). Furthermore, unlike GFP, thebioorthogonal chemical reporters are based on small molecules, which areless likely to perturb the functioning of the cell.

Bioorthogonal reactions via click chemistry are highly sensitive withlow background noise despite the complexities of the cellularenvironment. In practice, however, the sensitivity is constrained by theabundance of the target molecules, the labeling efficiency of thechemical reporters, and the performance of the exogenous probes. Inalmost all cases, bright and photostable probes are highly desirable,particularly for long-term tracking and sensitive detection oflow-abundance biomolecules.

Fluorescent nanoparticles have attracted much attention in recent years.The popular quantum dots (Qdots) exhibit improved brightness andphotostability over traditional fluorescent dyes. In the context ofclick chemistry, however, the copper catalyst irreversibly quenches Qdotfluorescence and prevents their usage in the various applications basedon copper-catalyzed click chemistry (S. Han, N. K. Devaraj, J. Lee, S.A. Hilderbrand, R. Weissleder, M. G. Bawendi, J. Am. Chem. Soc. 2010,132, 7838). Because of copper's cytotoxicity, copper-free bioorthogonalapproaches, such as the Staudinger ligation and the strain-promotedazide-alkyne cycloaddition, have been developed for live cell and invivo applications (Agard et al., supra), although these reactions cansometimes be more difficult to implement due to tedious syntheses (Hanet al., supra). Qdots can be employed in the copper-free methods (A.Bernardin, A. Cazet, L. Guyon, P. Delannoy, F. Vinet, D. Bonnaffe, I.Texier, Bioconjug. Chem. 2010, 21, 583), where their instability causedby copper is not an issue. However, Qdots' intrinsic toxicity, caused bythe leaching of heavy metal ions, is still a critical concern.

Semiconducting polymer dots (Pdots) represent a new class of ultrabrightfluorescent probes, which can overcome both issues for clickchemistry-based applications. Previous studies showed that Pdots werenot cytotoxic in different cellular assays, making them appealing forstudies in living system. In one aspect, the present invention isfocused on Pdot biological applications involving click chemistry. Pdotsare much brighter fluorescent probes than Qdots; have a thousand-foldfaster emission rates than Qdots; and are photostable and do not“blink”. For biological applications, however, a significant problem ofPdots is the control over their surface chemistry and conjugation tobiological molecules. This is a significant challenge that has preventedthe widespread adoption of Pdots in biological studies.

In one aspect, the present invention provides a general method thatovercomes this challenge by creating functional groups on the Pdotsurface. Because the formation of Pdot is driven by hydrophobicinteraction, some amphiphilic polymer with hydrophilic functional groupsmay be co-condensed into a single dot during nanoparticle formation. Thehydrophilic groups on the amphiphilic polymer can be used as handles forfunctionalizing the Pdots for conjugation to biomolecules. It was foundthat a general copolymer, poly(styrene-co-maleic anhydride) (PSMA),successfully functionalized the Pdots for further surface conjugations(FIG. 14). PSMA provides excellent options for Pdot functionalizationbecause it is commercially available in a broad range of molecularweights and maleic anhydride contents.

As shown in Example 17, PSMA was employed to functionalize Pdots madefrom a highly fluorescent semiconducting polymerPoly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,1′-3}-thiadiazole)](PFBT), but the method can be applied to any hydrophobic, fluorescent,semiconducting polymers. During Pdot formation, the hydrophobicpolystyrene units of PSMA molecules were likely anchored inside the Pdotparticles while the maleic anhydride units localized to the Pdot surfaceand hydrolyzed in the aqueous environment to generate carboxyl groups onthe Pdot surface. As shown herein, the carboxyl groups enable furthersurface conjugations.

As shown in Example 21, very low Pdot concentrations (˜50 nM), which wasorders of magnitude less than the general concentration used for smalldye molecules (typically in the μM range) can be used for biologicallabeling applications. These results also shown that Pdot tagging viaclick chemistry is highly specific, with virtually no backgroundlabeling in all the control samples. Additionally, the Pdot probes werealso used to detect glycoproteins and newly synthesized proteins detectin a different cell line, 3T3 fibroblast. In all these cases, the Pdotsalso specifically and effectively labeled the targets, demonstrating thestrategy was equally efficient and successful in different cell lines.

Accordingly, in one embodiment, the present invention provides a methodfor conjugation that covalently links functional molecules to Pdots forclick chemistry-based bioorthogonal labeling of cellular targets. Thesefunctionalized Pdots can selectively target, for example, newlysynthesized proteins and glycoproteins in mammalian cells that weremetabolically labeled with bioorthogonal chemical reporters. The highlyefficient, specific, and bright protein labeling using Pdots and clickchemistry demonstrate the potential of this method for visualizingvarious cellular processes. The methods and compositions described herewill enable Pdots to be used in a wide range of cellular studies andfluorescence applications.

In one embodiment, the present invention provides chromophoric polymerdot conjugates in which a molecule, preferably a biomolecule, isattached to the polymer dot by chemical bonding via a click reaction. Inorder to achieve this, a first functional group capable of participatingin a click chemistry reaction is engineered into the molecule (e.g., abiomolecule). For example, an attractive approach for installing azidesinto biomolecules is based on metabolic labeling, whereby anazide-containing biosynthetic precursor is incorporated intobiomolecules by using the cells' biosynthetic machinery. Then, a second,complementary functional group capable of participating in a clickchemistry reaction with the first functional group is attached to achromophoric polymer dot, as provided herein. Reactions between thefirst functional group in the biomolecule and the complementaryfunctional group on the polymer dot via click chemistry result in theformation of a polymer dot bioconjugate.

In one embodiment, an organic azide is used as functional group linkedor enriched in the biomolecule. A terminal alkyne as complementaryfunctional group is associated with polymer dot. As shown in FIG. 22(reaction i), the [3+2] cycicoaddition between azide and alkyne groupswith copper (I) as a catalyst lead to the formation of stable triazolelinkage between the polymer dot and the biomolecule. Alternatively,alkynes can be activated by ring strain. For example, cyclooctynes andtheir derivatives can react with azides at room temperature without theneed for a catalyst. The bioconjugation via the strain-promoted [3+2]cycloaddition (FIG. 22, reaction ii) removes the requirement forcytotoxic copper, which is particularly suitable for live cell and invivo applications. In another embodiment, biomolecule can be conjugatedto polymer dot via Staudinger ligation (FIG. 22, reaction iii). The termof “Staudinger ligation” is recognized in the art, which describes theselective reaction between phosphines and azides to form an amide bond.A functional phosphine group is associated with or covalently attachedto polymer dot. Reaction of the phosphine group with the azide group inthe biomolecule also results in formation of polymer dot bioconjugatewithout the need of catalyst. Click chemistry and Staudinger ligationcan be employed to conjugate free biomolecules to polymer dot. Theazide-, cyclooctyne-, and phosphine-functionalized polymer dots can alsobe used to specifically targeting the azide-containing biomolecules invitro or in vivo.

Accordingly, in one embodiment, the present invention provides afunctionalized chromophoric polymer dot (Pdot) comprising a core and acap, wherein said core comprises a chromophoric polymer, and said capcomprises a functionalization agent bearing one or more functionalgroups capable of being conjugated to a molecule via a click chemistryreaction, with the proviso that not all of the said cap is an organosilicate. In a specific embodiment, the functional group is selectedfrom an alkyne, strained alkyne, azide, diene, alkene, tetrazine,strained alkene, cyclooctyne, phosphine groups, and other groups forclick reaction and other bioorthogonal reactions.

In a related embodiment, the present invention provides a functionalizedchromophoric polymer dot (Pdot) having a hydrophobic core and ahydrophilic cap, the Pdot comprising: (a) a semiconducting polymer; and(b) an amphiphilic molecule, having a hydrophilic moiety and ahydrophobic moiety, attached to a reactive functional group capable ofbeing conjugated to a molecule via a click chemistry reaction, whereinthe semiconducting polymer is embedded within the hydrophobic core ofthe Pdot and; wherein a portion of the amphiphilic molecule is embeddedwithin the core of the Pdot and the reactive functional group is locatedin the hydrophilic cap. In a specific embodiment, the functional groupis selected from an alkyne, strained alkyne, azide, diene, alkene,tetrazine, strained alkene, cyclooctyne, phosphine groups, and othergroups for click reaction and other bioorthogonal reactions.

Bioconjugated Chromophoric Polymer Dots

In another aspect, the present invention provides a bioconjugatecomprising a functionalized chromophoric polymer dot as described hereinand a biomolecule, wherein the biomolecule is attached to the polymerdot either directly or indirectly by any suitable means. Thebioconjugates also comprise functionalized chromophoric polymer dot asdescribed above, physically or chemically associated with biologicalparticle such as virus, cells, biological or synthetic vesicles such asliposomes.

The term “biomolecule” is used to describe a synthetic or naturallyoccurring protein, glycoprotein, peptide, amino acid, metabolite, drug,toxin, nuclear acid, nucleotide, carbohydrate, sugar, lipid, fatty acidand the like. The biomolecule may be attached to the polymer dotdirectly or indirectly by any suitable means, such as by any stablephysical or chemical association. Desirably, the biomolecule is attachedto the hydrophilic functional groups of the functionalized polymer dotvia one or more covalent bonds. For example, if the functional group ofthe polymer dot is carboxyl group, a protein biomolecule can be directlyattached to the polymer dot by cross-linking the carboxyl group withamine group of the protein biomolecule.

The term “cross-linking agent” is used to describe a compound that iscapable of forming a chemical bond between molecular groups on similaror dissimilar molecules so as to covalently bond together the molecules.In the present invention, a suitable cross-linking agent is one thatcouple carboxyl to amine groups, for example1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). Otherexamples of common cross-Linking agents are known in the art. See, forexample, Bioconjugate Techniques (Academic Press, New York, 1996 orlater versions). Indirect attachment of the biomolecule to the˜hromophoric polymer dots can occur through the use of “linker”molecule, for example, avidin streptavidin, neutravidin, biotin or alike molecule.

Accordingly, in one embodiment, the present invention provides afunctionalized thromophoric polymer dot having a hydrophobic core and ahydrophilic cap, as provided nerein, wherein a biological molecule isconjugated to a reactive functional group present in the hydrophilic capstructure (i.e., on the surface of the polymer dot).

Generally, any biological molecule of interest may be conjugated to aclu-omophoric polymer dot provided herein, including without limitationtargeting molecules, effector molecules, inhibitor molecules, imagingmolecules, and the like. Exemplary biological molecules that may beconjugated to a chromophoric polymer dot provided by the inventioninclude, synthetic or naturally occurring proteins, glycoproteins,polypeptides, amino acids, nucleic acids, carbohydrates, lipids, fattyacids, and combinations thereof.

In a specific embodiment, the present invention provides afunctionalized chromophoric polymer dot (Pdot) comprising a core and acap, wherein said core comprises a chromophoric polymer, and said capcomprises a functionalization agent bearing one or more functionalgroups conjugated to a biological molecule, with the proviso that notall of the said cap is an organo silicate. In one embodiment, thebiological molecule is selected from a synthetic protein, a naturallyoccurring protein, a glycoprotein, a polypeptide, an amino acid, anucleic acid, a carbohydrate, a lipid, a fatty acid, and a combinationthereof. In a specific embodiment the biological molecule is apolypeptide. In one embodiment, the polypeptide is an antibody orfragment thereof. In another specific embodiment, the biologicalmolecule is a nucleic acid. In one embodiment, the biological moleculeis an aptamer.

In a related aspect, the present invention provides a functionalizedchromophoric polymer dot (Pdot) having a hydrophobic core and ahydrophilic cap, the Pdot comprising: (a) a semiconducting polymer; and(b) an amphiphilic molecule bearing a reactive functional groupconjugated to a biological molecule, wherein the semiconducting polymeris embedded within the hydrophobic core of the Pdot and; wherein aportion of the amphiphilic molecule is embedded within the core of thePdot and the reactive functional group and conjugated biologicalmolecule is located in the hydrophilic cap (i.e., on the surface of thepolymer dot).

In one embodiment, the reactive functional group used to conjugate abiological molecule to a chromophoric polymer dot, is a chemical moietycapable of participating in a click chemistry reaction and otherbioorthogonal reactions. In a specific embodiment, the functional groupis selected from an alkyne containing moiety, an azido containingmoiety, and a phosphine containing moiety.

In some embodiments, the chromophoric polymer dot is conjugated to an“effector moiety”. The effector moiety can be any number of molecules,including labeling moieties such as radioactive labels or fluorescentlabels; targeting moieties such as antibodies, aptamers, proteins,peptides; or can be a therapeutic moiety. Such therapeutic moietiesinclude, but are not limited to, an anti-tumor drug, a toxin, aradioactive agent, a cytokine, an antibody or an enzyme. Further, theinvention provides an embodiment wherein the chromophoric polymer dot ofthe invention is linked to an enzyme that converts a prodrug into acytotoxic agent.

In yet other embodiments, a functionalized chromophoric polymer dot, asprovided herein, is conjugated to more than one type of effectormolecule. For example, in one embodiment, a chromophoric polymer dot isconjugated to both a targeting moiety (e.g., an antibody or aptamer) anda therapeutic moiety (e.g., an anti-tumor drug, a toxin, a radioactiveagent, a cytokine, an antibody or an enzyme). As such, thefunctionalized chromophoric polymer dots provided herein may serve as aboth a diagnostic/imaging tool, through the fluorescence of the polymerdot itself, and a therapeutic moiety.

Chromophoric Polymer Dots for In Vivo Applications

Nanoparticle-based diagnostic and therapeutic agents have attractedconsiderable interest because of their potential for clinical oncologyand other biomedical research. Versatile nanostructures have beendemonstrated for in vivo applications, such as lipid and polymericnanocapsules for drug delivery, iron oxide nanoparticles for magneticresonance imaging, gold nanoparticles for X-ray computed tomography, andquantum dots (Qdots) for fluorescence imaging. Among those, the organicmolecule based nanocapsules provide flexible vehicles for drugencapsulation and delivery. However, they rarely provide imagingcontrast, and generally require a molecular tag to enable in vivomonitoring by fluorescence. Conversely, inorganic nanoparticles areprimarily used as imaging contrast agents owing to their uniqueproperties. Qdots represent one of the exciting nanotechnologiestranslated to biology in the past decade. The size-tunable luminescencemake them appealing as multicolor fluorophores for extensive biologicallabeling, imaging, and sensing. For in vivo applications, however, theintrinsic toxicity of Qdots is of critical concern, which may impedetheir final clinical translation.

In a recent landmark paper (Choi, H. S. et al. Nature Biotechnol. 25,1165-1170 (2007)), three criteria were proposed for distinguishing ananoparticle that has potential clinical utility: (i) a finalhydrodynamic diameter (HD)<5.5 nm to permit fast renal clearance and/or(ii) a formulation with completely nontoxic components and/or (iii)biodegradability to clearable components. Although small HD (<5.5 nm)could result in efficient renal clearance to mitigate toxic effect(Id.), such a size limit poses great challenges for most nanoparticlesystems, particularly the quantum dots (Choi, H. S. et al. NatureNanotechnol. 5, 42-47 (2010)), where the luminescence is size-dependentand an encapsulation layer is required for water solubility. Therefore,design of bright fluorescent probes with biologically benign materialsis highly desirable for many in vivo applications related to diagnosisand treatment of human disease.

Semiconducting polymers combine the organic polymeric nature with theunique optical properties of semiconductors. The motivation of adaptingsemiconducting polymers as fluorescent nanoparticle labels stems from anumber of favorable characteristics such as the high per-particlebrightness, fast emission rates, and excellent photostability.Furthermore, semiconducting polymer dots (Pdots) are intrinsicallybenign and biocompatible: cytotoxicity was not observed in differentcell lines incubated with highly concentrated nanoparticles for days(Moon, J. H. et al., Angew. Chem. Int. Ed. 46, 8223-8225 (2007); and Pu,K. Y. et al., Chemistry of Materials 21, 3816-3822 (2009)). While stillat the early stage of development, Pdots have been attracting intensiveinterest (see, for example, Pecher, J. & Mecking, S. Chem. Rev. ArticleASAP (2010); and Kaeser, A. & Schenning, A. P. H. J. Adv. Mater. 22,2985-2997 (2010)).

Researchers including synthetic chemists have developed various methodsto improve the nanoparticle's versatility and functions for biomedicalstudies, such as tuning the emission color (Abbel, R., et al., Chem.Commun., 1697-1699 (2009), the disclosure of which is hereinincorporated by reference in its entirety for all purposes), exploringnew preparation methods (Baier, M. C. et al., J. Am. Chem. Soc. 131,14267-14273 (2009), the disclosure of which is herein incorporated byreference in its entirety for all purposes), engineering the particlesurface (Howes, P. et al., J. Am. Chem. Soc. 132, 3989-3996 (2010), thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes), encapsulating magnetic materials (Howes, P. et al. J.Am. Chem. Soc. 132, 9833-9842 (2010), the disclosure of which is hereinincorporated by reference in its entirety for all purposes), and thefirst in vivo experiment for sentinel lymph node mapping (Kim, S. et al.Chem. Commun. 46, 1617-1619 (2010), the disclosure of which is hereinincorporated by reference in its entirety for all purposes). Despite allthe efforts, it is still unclear whether these polymer based novelprobes can be specifically targeted to malignant tumors in vivo.Specific tumor targeting is the first prerequisite for any furtherclinical considerations. In one aspect, the present invention overcomesseveral challenges related to ligand conjugation and probe performanceof chromophoric nanoparticles useful for in vivo application.

As described herein, various semiconducting polymers can be used forpreparing small Pdots as fluorescent labels. Particularly, polyflourenes(PF) and their derivatives exhibit high fluorescence quantum yields andexcellent thermal and chemical stability. Significant success has beenmade to tune their emission color from blue to deep-red region byintroducing narrow-band-gap monomers into the polymer backbone (see,Hou, Q. et al. Macromol. 37, 6299-6305 (2004); and Yang, R. Q. et al.Macromol. 38, 244-253 (2005), the disclosures of which are hereinincorporated by reference in their entireties for all purposes),exhibiting great flexibility for designing fluorescent probes. However,the fluorescence quantum yield, particularly in the deep-red region,drops significantly as the concentration of narrow-band-gap monomers isincreased (see, Id.). As a trade off, only a small amount ofnarrow-band-gap monomers were incorporated in the PF copolymer tomaintain a high fluorescent quantum yield, which resulted in thedominant absorption of relative Pdots in the ultraviolet (UV) region(FIG. 23). This is a severe drawback for in vivo applications.

In order to overcome the above limitation, the present inventionprovides nanoparticles consisting of donor-acceptor polymer blends,which take advantage of the efficient intra-particle energy transferthat occurs in Pdots (see, for example, Wu, C. et al., J. Phys. Chem. C112, 1772-1781 (2008); and Wu, C. et al., J. Phys. Chem. B 110,14148-14154 (2006)). The polymer blend dots (“PBdots”) were prepared byusing a visible-light-harvesting polymer (PFBT) as donor and anefficient deep-red emitting polymer (PF-0.1TBT) as acceptor (polymerstructures shown in FIG. 24). Because the donor and acceptor polymerswere closely packed into single dots, intra-particle energy transferresulted in complete quenching of the PFBT donor, accompanied by aneffective fluorescence from the acceptor polymer alone (FIG. 25B). At agiven blending ratio of 0.6 (PF-0.1TBT to PFBT in weight), the PBdotsexhibit a broad visible absorption band extending to 600 nm, and anefficient 650 nm emission with a quantum yield of 0.52 (FIGS. 25C and26A).

The blending strategy was also successfully applied to other polymerdonor-acceptor systems consisting of light-harvesting polymer PFPV anddifferent red-emitting polymers (FIG. 27), indicating its generalapplication for tuning Pdot properties. Although Pdots emitting in nearinfrared (NIR) region are more preferable for in vivo imaging, as shownin Example 29, the current PBdots represent the brightest probe indeep-red region among various nanoparticles of similar size (˜15 nmdiameter), which may significantly overcome background autofluorescenceand scattering in biological tissues.

Accordingly, in one embodiment, the present invention provides achromophoric polymer dot having a deep-red-shifted emission. In oneembodiment, the chromophoric polymer dot comprises a blend of apolyflourene polymer (PF) and fluorene-benzothiadiazole-thiophencopolymer (PF-0.1TBT). The narrow-band-gap TBT monomers were introducedinto polyfluorene backbone in order to tune the emission color from blueto deep-red region. (see, Hou, Q. et al. Macromol. 37, 6299-6305 (2004);and Yang, R. Q. et al. Macromol. 38, 244-253 (2005)). The concentrationof TBT monomers can be varied from 0.01 to 0.5. Advantageously, thesepolymer blends comprising PFBT and PF-0.1TBT result in dominantabsorption in the visible region and deep-red fluorescence withoutsignificant loss of quantum yield. Generally, the blend will containbetween about 2% and about 75% PF-0.1TBT copolymer. In certainembodiment, the ratio of PF to PF-0.1TBT is no greater than about 50:1or no greater than about 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1,17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, 3:2, 1:1, 2:3, 1:2, or 1:3. In one specific embodiment,the polymer blends comprise PFBT and PF-0.1TBT. In another specificembodiment, the polymer blends comprise PFPV and PF-0.1TBT. In apreferred embodiment, the polymer dot comprises a functional group onits surface.

In a specific embodiment, the deep-red shifted polymer dot, having ahydrophobic core and a hydrophilic cap, comprises: (a) a blend of a PFBTand PF-0.1TBT semiconducting polymers; and (b) an amphiphilic molecule,having a hydrophilic moiety and a hydrophobic moiety, attached to areactive functional group, wherein the semiconducting polymers areembedded within the hydrophobic core of the Pdot and; wherein a portionof the amphiphilic molecule is embedded within the core of the Pdot andthe reactive functional group is located in the hydrophilic cap (i.e.,on the surface of the polymer dot). In some embodiments, the polymer dotis further conjugated to an effector molecule (e.g., a targeting agent).In one embodiment, the red shifted polymer dot has a peak emissionbetween about 600 nm and about 700 nm (i.e., in the deep red region).

In another embodiment, the deep-red shifted polymer dot, having ahydrophobic core and a hydrophilic cap, comprises: (a) a PFBT polymer;and (b) a PF-0.1TBT semiconducting polymer, wherein a sub-population ofthe semiconducting polymers harbors a reactive functional group, whereinthe semiconducting polymers not harboring reactive functional groups areembedded within the hydrophobic core of the Pdot and; wherein a portionof the semiconducting polymers harboring reactive functional groups areembedded within the core of the Pdot and the reactive functional groupis located in the hydrophilic cap (i.e., on the surface of the polymerdot). In some embodiments, the polymer dot is further conjugated to aneffector molecule (e.g., a targeting agent).

In a related embodiment, deep-red shifted polymer dots are providedwhich are conjugated to an effector molecule, for example, a targetingmoiety. In one embodiment, the bioconjugated chromophoric polymer dotcomprises a blend of a semiconducting polymer (e.g., PF, PFBT, PFPV,etc.) and PF-0.1TBT semiconducting polymer. Generally, the blend willcontain between about 2% and about 75% PF-0.1TBT copolymer. In certainembodiment, the ratio of PF to PF-0.1TBT is no greater than about 50:1or no greater than about 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1,17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, 3:2, 1:1, 2:3, 1:2, or 1:3. In one specific embodimentthe polymer blends comprise PFBT and PF-0.1TBT. In another specificembodiment, the polymer blends comprise PFPV and PF-0.1TBT.

In another aspect, the present invention provides chromophoric polymerdots having near-infrared (NIR) fluorescent emission. As used herein,“near-infrared emission” refers to electromagnetic radiation having awavelength between about 700 nm and about 1500 nm. As shown in Example29, NIR fluorescent emission can be achieved by embedding a NIR dye intothe hydrophobic core of a chromophoric polymer dot provided herein. ManyNIR fluorescent dyes are known in the art, for example cyanine dyes arethe most commonly used near-IR fluorescent dyes. Other NIR dye familiesinclude oxazine, rhodamine, and phthalocyanine dyes. To achieve NIRemission after excitation, the NIR Pdot takes advantage of efficientintermolecular FRET between the chromophoric polymer comprising the Pdotand the NIR dye embedded within the hydrophobic core of the molecule.Accordingly, when selecting a NIR dye to be used for the preparation ofa NIR Pdot, care must be taken to select chromophores having overlappingemission and excitation spectras (i.e., an appropriate chromophoricpolymer FRET donator and NIR dye FRET acceptor). In a preferredembodiment, the NIR polymer dot is further functionalized on itssurface.

Accordingly, in one embodiment, a NIR polymer dot comprises a core and acap, wherein said core comprises a chromophoric polymer and a NIR dye,and said cap comprises a functionalization agent bearing one or morefunctional groups, with the proviso that not all of said cap is anorgano silicate. In one embodiment, the polymer dot is furtherconjugated to an effector molecule (e.g., a targeting agent).

In a related embodiment, a NIR polymer dot, having a hydrophobic coreand a hydrophilic cap, comprises: (a) a semiconducting polymer; (b) anNIR dye; and (c) an amphiphilic molecule, having a hydrophobic moietyand a hydrophilic moiety attached to a reactive functional group,wherein the semiconducting polymer and NIR dye are embedded within thehydrophobic core of the Pdot and; wherein a portion of the amphiphilicmolecule is embedded within the core of the Pdot and the reactivefunctional group is located in the hydrophilic cap. In one embodiment,the polymer dot is further conjugated to an effector molecule (e.g., atargeting agent).

Methods for Preparing Chromophoric Polymer Dots

In one aspect, the present invention provides novel methods forfunctionalizing the surface of chromophoric nanoparticles (i.e.,chromophoric polymer dots or “Pdots”). In one embodiment, the method isbased on a concept in which heterogeneous polymer chains are entrappedinto a single Pdot by hydrophobic interactions established duringnanoparticle formation. A small amount of amphiphilic polymers isco-condensed with the semiconducting polymers of the Pdots to modify andfunctionalize the nanoparticle surface.

FIG. 1 illustrates an example of this, by using a functional,amphiphilic, comb-like, polystyrene polymer PS-PEG-COOH, however, otheramphiphilic polymers with different functional groups can also be used.PS-PEG-COOH consists of a hydrophobic polystyrene backbone and severalhydrophilic side chains of ethylene oxide terminated with carboxylicacid. During nanoparticle formation, the hydrophobic polystyrenebackbones are embedded inside the Pdot particles while the hydrophilicPEG chains and functional groups extend outside into the aqueousenvironment. Unlike physical adsorption, therefore, this method shouldpermanently anchors the PEG and functional groups to the Pdot surface.The PEG chains provide a biocompatible layer that minimizes non-specificabsorption. The PEG chains also act as a steric barrier againstnanoparticle aggregation, while the functional carboxyl group can beeasily covalently-linked to biomolecules using established protocols(see, for example, Xing, Y.; Chaudry, Q.; Shen, C.; Kong, K. Y.; Zhau,H. E.; WChung, L.; Petros, J. A.; O'Regan, R. M.; Yezhelyev, M. V.;Simons, J. W.; Wang, M. D.; Nie, S. Nature Protoc. 2007, 2, 1152-1165,the contents of which is herein incorporated by reference in itsentirety for all purposes).

In a preferred embodiment, chromophoric polymer dots are prepared byprecipitation. This technique involves the rapid addition (e.g.,facilitated by sonication or vigorous stirring) of a dilute chromophoricpolymer solution (e.g., chromophoric polymer dissolved in an organicsolvent) into an excess volume of non-solvent (but miscible with theorganic solvent), such as water or another physiologically relevantaqueous solution. For example, in some embodiments, the hydrophobicchromophoric polymer is first dissolved into an organic solvent wherethe solubility is good (good solvent), such as THF (tetrahydrofuran),after which the dissolved polymer in THF is added to an excess volume ofwater or buffer solution, which is a poor solvent for the hydrophobicchromophoric polymers but which is miscible with the good solvent (THF).The resulting mixture is sonicated or vigorously stirred to assist theformation of chromophoric polymer dots, then the organic solvent isremoved to leave behind well dispersed chromophoric nanoparticles. Inusing this procedure, the chromophoric polymer must be sufficientlyhydrophobic to dissolve into the organic solvent (e.g. THF).

However, other methods of forming chromophoric polymer dots are alsopossible, including but not limited to various methods based onemulsions (e.g., mini or micro emulsion) or precipitations orcondensations. In one embodiment, hydrophobic functional groups may belocalized to the surface of the polymer dot such that they will beavailable for conjugation, for example, to a biomolecule. In one scheme,hydrophobic reactive functional groups are localized to the surface ofthe Pdot by attaching the functional group to a hydrophilic linker thatcarries the functional group to the hydrophilic exterior of the polymerdot. This latter approach may work particularly well using functionalgroups.that are both hydrophobic and clickable (i.e., chemical reactionsthat fall within the framework of click chemistry), including but notlimited to alkyne, strained alkyne, azide, diene, alkene, cyclooctyne,and phosphine groups.

Accordingly, in a preferred embodiment, the present invention provides amethod for preparing a functionalized chromophoric polymer dot byintroducing a mixture of a semiconducting polymer and an amphiphilicmolecule attached to a reactive functional group solvated in anon-protic solvent into a solution comprising a protic solvent. In oneembodiment, the method further comprises filtering the suspension toisolate a population of polymer dots of a particular size.

In a specific embodiment, the method for preparing a functionalizedchromophoric polymer dot comprises the steps of (a) preparing a mixtureof a semiconducting polymer and an amphiphilic molecule attached to areactive functional group in a non-protic solvent; (b) introducing(e.g., by injecting) all or a portion of the mixture into a solutioncomprising a protic solvent, thereby collapsing the semiconductingpolymer and amphiphilic molecule into a nanoparticle; and (c) removingthe aprotic solvent from the mixture formed in step (b), thereby forminga suspension of functionalized chromophoric polymer dots, wherein aportion of the amphiphilic molecule is embedded within the core of thenanoparticle and the reactive functional group is located on the surfaceof the nanoparticle. In one embodiment, the method further comprisesfiltering the suspension to isolate a population of polymer dots of aparticular size.

In one specific embodiment, the amphiphilic polymer is furtherconjugated to an effector molecule through a reactive functional groupprior to introducing the mixture into the solution comprising a proticsolvent. This approach will work for molecules that tolerate non-proticsolvents, but won't work well with other molecules (e.g., proteins) thatdo not. As a demonstration of the feasibility of this strategy, Example31 provides a case wherein an amphiphilic polymer containing a reactivefunctional group was first conjugated to a pH sensitive fluoresceinisothiocyanate dye and then blended with a semiconducting polyer to forma conjugated polymer dot.

Many techniques are know in the art for removing an aprotic solvent froma mixture comprising an aprotic and a protic solvent, including withoutlimitation, distillation, nitrogen stripping, and variouschromatographic techniques (e.g., buffer exchange chromatography). In apreferred embodiment, wherein the boiling point of the aprotic solventis lower than the boiling point of the protic solvent, the aproticsolvent is removed from the mixture formed in step (b) by nitrogenstripping. In a preferred embodiment, the protic solvent is water.

Methods for Preparing Chromophoric Polymer Dot Bioconjugates

As described herein, the functionalized chromophoric polymer dotsprovided by the present invention allow for the facile conjugation ofbiological molecules to stable, non-toxic fluorescent probes (i.e.,Pdots) that can be used in a wide array of diagnostic and experimentalassays. In this fashion, a molecule, preferably a biomolecule, may beattached to a chromophoric polymer dot through adsorption to the surfaceof the polymer dot (e.g., mediated through electrostatic or hydrophobicinteractions) or by direct chemical attachment.

As used herein, a “bioconjugated CPdot” or “bioconjugated Pdot” refersto a chromophoric polymer dot with any biomolecule stably attachedthrough any stable physical or chemical association. “Bioconjugation” inthis application refers to a process that conjugates biomolecules to thechromophoric polymer dot by any stable physical or chemical association.

1. Bioconjugation Through Physical Adsorption

A biomolecule is stably associated with the chromophoric polymer dots byphysical adsorption. Physical adsorption can arise from a range offorces, including but not limited to van de Waals, electrostatic,pi-stacking, hydrophobic, entropic forces and combinations thereof.Physical adsorption will be mediated by the physical and chemicalproperties of the cap structure on the chromophoric polymer dot and thetarget molecule (e.g., biomolecule) being adsorbed. As such, thefunctional group attached to the polymer dot through the hydrophilic capstructure, will determine the type of molecules that may be adsorbed onto the polymer dot.

Accordingly, in one embodiment, the present invention provides abioconjugated chromophoric polymer dot comprising a hydrophobic core anda hydrophilic cap, wherein said core comprises a chromophoric polymer,and said cap comprises a functionalization agent bearing one or morefunctional groups to which a biological molecule is adsorbed.

In one embodiment, a bioconjugated chromophoric polymer dot is preparedby introducing a mixture of a semiconducting polymer and an amphiphilicmolecule attached to a reactive functional group solvated in anon-protic solvent into a solution comprising a protic solvent toprepare a functionalized chromophoric polymer dot and then adsorbing abiological molecule onto the surface of the polymer dot. In oneembodiment, the method further comprises filtering the suspension toisolate a population of polymer dots of a particular size prior toadsorbing the biological molecule onto the polymer dot.

In a specific embodiment, the method for preparing a bioconjugatedchromophoric polymer dot comprises the steps of (a) preparing a mixtureof a semiconducting polymer and an amphiphilic molecule attached to areactive functional group in a non-protic solvent; (b) introducing(e.g., by injecting) all or a portion of the mixture into a solutioncomprising a protic solvent, thereby collapsing the semiconductingpolymer and amphiphilic molecule into a nanoparticle; (c) removing theaprotic solvent from the mixture formed in step (b), thereby forming asuspension of functionalized chromophoric polymer dots, wherein aportion of the amphiphilic molecule is embedded within the core of thenanoparticle and the reactive functional group is located on the surfaceof the nanoparticles; and (d) adsorbing a biomolecule of interest ontothe surface. In one embodiment, the method further comprises filteringthe suspension to isolate a population of polymer dots of a particularsize.

2. Bioconjugation Through Chemical Bonding

In a preferred embodiment, a molecule (e.g., a biomolecule) is attachedto the polymer dot by chemical bonding, which requires that functionalgroups be available on the polymer dot, such as carboxylic acid, amino,mercapto, azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate,sulfonate, phosphate, cyanate, succinimidyl ester, substitutedderivatives thereof, or combinations there of. In general, anyfunctional groups that allow bioconjugation may be used. Such groupscould be found by one of ordinary skill in the art, for example inBioconjugate Techniques (Academic Press, New York, 1996 or secondedition, 2008; the disclosures of which are herein incorporated byreference in their entireties for all purposes). Then the bioconjugationcan be done by standard bioconjugation techniques. (Id.)

Accordingly, in one embodiment, the present invention provides abioconjugated chromophoric polymer dot comprising a core and a cap,wherein said core comprises a chromophoric polymer, and said capcomprises a functionalization agent bearing one or more functionalgroups to which a biological molecule is covalently attached.

In a specific embodiment, the present invention provides a bioconjugatedchromophoric polymer dot having a hydrophobic core and a hydrophiliccap, the polymer dot comprising: (a) a chromophoric polymer; and (b) anamphiphilic molecule conjugated to a biological molecule via a reactivefunctional group, wherein the chromophoric polymer is embedded withinthe hydrophobic core of the Pdot and; wherein a portion of theamphiphilic molecule is embedded within the core of the Pdot and theconjugated biological molecule is located in the hydrophilic cap (i.e.,on the surface of the polymer dot). In a preferred embodiment, thechromophoric polymer is a semiconducting polymer.

2a. Covalent Modification of Chromophoric Polymers

One synthetic strategy for making functionalized CPdot is a two-stepprocess: the first step is to synthesize a chromophoric polymer bearingfunctional groups such as carboxylic acid, amino, mercapto, azido,alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate, phosphate,cyanate, succinimidyl ester, substituted derivatives thereof, orcombinations there of. In general, any functional groups that allowbioconjugation may be used. Such groups could be found by one ofordinary skill in the art, for example in Bioconjugate Techniques(Academic Press, New York, 1996 or second edition, 2008). Functionalgroups can be created with covalent bonding to the backbone or a sidechain of the chromophoric polymer. Such chemical modification is knownto one of skill in the art. In the second step, the functionalizedchromophoric polymer is used as a precursor for preparing CPdots (thatwould have functional groups available for bioconjugation). The CPdotpreparation from the functionalized chromophoric polymer can use themethod provided in this application (Example 1), based on mixing twomiscible solvents. The CPdot preparation from the functionalizedchromophoric polymer can also be achieved by a emulsion or miniemulsionmethod, based on shearing a mixture comprising two immiscible liquidphases (such as water and another immiscible organic solvent) in thepresence of a surfactant. A shearing process such as ultrasonicationmakes stable droplets that contain the functionalized chromophoricpolymers in the suspension. After removing the organic solvent,functionalized CPdots are obtained, which typically have a size from afew nanometer to sub-microns.

Accordingly, in one embodiment, the present invention provides a methodfor preparing a bioconjugated chromophoric polymer dot, the methodcomprising forming a chromophoric polymer dot having a hydrophobic coreand a hydrophilic cap with a chromophoric polymer bearing one or morereactive functional groups, wherein the reactive functional groups arelocalized in the hydrophilic cap (i.e., on the surface of the polymerdot) and covalently attaching a biomolecule to the polymer dot via alinkage to the reactive functional group.

Another synthetic strategy of making functionalized CPdots may bedemonstrated in a process combining the synthesis of chromophoricpolymer with nanoparticle formation. A portion of the monomer unitsand/or the terminating units for synthesizing the chromophoric polymerhave functional groups such as carboxylic acid, amino, mercapto, azido,alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate, phosphate,cyanate, succinimidyl ester, substituted derivatives thereof, orcombinations there of. In general, any functional groups that allowbioconjugation may be used. Such groups could be found by one ofordinary skill in the art, for example in Bioconjugate Techniques(Academic Press, New York, 1996 or later versions). Formed by emulsionor other methods, droplets may serve as micro or nano-reactors thatcontain the monomers, functional monomers and catalyst for synthesizingthe chromophoric polymer dot. Chromophoric polymer dot obtained by thismethod are expect to show well controlled size from nanometer to micronsize and have surface functional groups such as carboxylic acid, amino,mercapto, azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate,sulfonate, phosphate, cyanate, succinimidyl ester, substitutedderivatives thereof, or combinations there of. Then the bioconjugationcan be done by standard bioconjugation techniques. [BioconjugateTechniques, Academic Press, New York, 1996]

2b. Blending of Functionalization Agents

Alternatively, the functionalized CPdots can be prepared by collapsingone or more chromophoric polymers (many of which are commerciallyavailable) in the presence of one or more amphiphilic functionalizationagents harboring a functional group. The amphiphilic nature of thefunctionalization agents allow the hydrophobic portion of the moleculeto be anchored in the hydrophobic core of the collapsed polymer dot,while the hydrophilic portion of the molecule, containing one or morefunctional groups, is localized to the hydrophilic cap of the polymerdot. In certain embodiments, the functionalization agent may be achromophoric polymer that has been functionalized. In other embodiments,the functionalization agent may be a non-chromophoric molecule.

Non-limiting examples of functionalization agents that may be used tofunctionalize the chromophoric polymer dots of the present inventioninclude, small organic molecules, lipid molecules, and polymer moleculesthat comprise functional groups such as carboxylic acid or saltsthereof, amino, mercapto, azido, alkyne, aldehyde, hydroxyl, carbonyl,sulfate, sulfonate, phosphate, cyanate, succinimidyl ester, substitutedderivatives thereof, or combinations there of. In general, anyfunctional groups that allow bioconjugation may be used. Such groupscould be found by one of ordinary skill in the art, for example inBioconjugate Techniques (Academic Press, New York, 1996 or secondedition, 2008). These molecules can be associated with the core ofchromophoric polymer dot by any chemical bonding and/or physical forces,and provide surface functional groups on the chromophoric polymer dotfor bioconjugation.

Preferably, the functionalization agent is an amphiphilic polymer thatcomprises a hydrophobic moiety and a hydrophilic moiety containingfunctional groups. The hydrophobic moiety is physically embedded in thepolymer dots by hydrophobic interactions, while the hydrophilic moietyextends into the solution. This physical embedding of the hydrophobicmoiety in the polymer dots may be further facilitated if desired by theuse of chemical association between the hydrophobic moiety of thefunctionalization agent and the polymer dot. More preferably, thefunctionalization agent is a comb-like amphiphilic polymer comprisingmultiple repeating units of the hydrophobic moiety and multiplerepeating units of the hydrophilic moiety so that the functionalizationagent is permanently anchored to the polymer dot by strong hydrophobicforces, and the hydrophilic moieties comprise functional groupsextending into the solution for bioconjugation.

The hydrophobic moiety of an amphiphilic functionalization molecule maybe, for example, an alkyl group, more preferably an aryl group tostrengthen the hydrophobic attachment to the chromophoric polymer dotthat consists of many aromatic rings. The hydrophilic moiety may be, forexample, a polyalkalene glycol or preferably polyethylene glycol, whichmay further be substituted by groups such as carboxylic acid, amino,mercapto, azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate,sulfonate, phosphate, cyanate, succinimidyl ester, substitutedderivatives thereof, or combinations there of. In general, anyfunctional groups that allow bioconjugation may be used. Such groupscould be found by one of ordinary skill in the art, for example inBioconjugate Techniques (Academic Press, New York, 1996 or secondedition, 2008).

The functionalized CPdots from the chromophoric polymer and afunctionalization agent can be prepared by the method provided in thisapplication (Example 1), based on mixing two miscible solvents. Thefunctionalized CPdots by the chromophoric polymer and afunctionalization agent can also be prepared by a emulsion orminiemulsion method, based on shearing a mixture comprising twoimmiscible liquid phases (such as water and another immiscible organicsolvent) with the presence of a surfactant. A shearing process such asultrasonication makes stable droplets that contain the chromophoricpolymer and functionalization agent. After removing the organic solvent,functionalized CPdots are obtained, which typically have a size from fewnanometer to sub-microns. These functionalized CPdots can be furtherbioconjugated.

Accordingly, in one embodiment, the present invention provides a methodfor preparing a bioconjugated chromophoric polymer dot by (i) forming afunctionalized chromophoric dot having a hydrophobic core and ahydrophilic cap, the Pdot comprising: (a) a chromophoric polymer; and(b) an amphiphilic molecule, having a hydrophobic moiety and ahydrophilic moiety attached to a reactive functional group, wherein thechromophoric polymer is embedded within the hydrophobic core of the Pdotand; wherein a portion of the amphiphilic molecule is embedded withinthe core of the Pdot and the reactive functional group is located in thehydrophilic cap; and (ii) covalently attaching a biomolecule to thepolymer dot via a linkage to the reactive functional group. In apreferred embodiment, the chromophoric polymer is a semiconductingpolymer.

2c. Reduction of Non-Specific Binding

As demonstrated in Example 11, significant non-specific adsorption ofbiomolecules can occur on the surface of functionalized Pdots. Thisnon-specific adsorption interferes with the covalent attachment oftargeted molecules, rendering the product of conjugation reactionsunusable. Advantageously, it was found that the addition of freepolyethylene glycol to the conjugation reaction eliminated non-specificbinding between the biological molecule of interest and thefunctionalized Pdots, allowing for completion of the conjugationreaction. This finding is demonstrated in Example 11 provided herein.

Accordingly, in one embodiment, the present invention provides a methodfor conjugating a biological molecule to a functionalized chromophoricpolymer dot, the method comprising incubating a functionalizedchromophoric polymer dot with the biological molecule in a solutioncontaining water soluble polymer under conditions suitable forconjugating the biological molecule to the functionalized chromophoricpolymer dot, wherein the presence of the water soluble polymer in thesolution reduces non-specific adsorption of the biological molecule tothe surface of the polymer dot. Generally, any water soluble polymerthat reduces the non-specific binding of a target biological moleculeand the surface of the polymer dot may be used. Non-limiting examples ofwater soluble polymers include, a polyalkylene glycol (e.g., a PEG), aPEO, a polypropylene glycol, a polyoxyalkylene, a starch, apoly-carbohydrate, a polysialic acid, and the like. In a preferredembodiment, the water soluble polymer is a polyalkylene glycol. In aspecific embodiment, the water soluble polymer is polyethylene glycol.

In one embodiment, the functionalized chromophoric polymer dotcomprises: (a) a semiconducting polymer; and (b) an amphiphilic moleculeattached to a reactive functional group, wherein a portion of theamphiphilic molecule is embedded within the core of the polymer dot andthe reactive functional group is located on the surface of the polymerdot.

In one embodiment, the present invention provides a method forconjugating a biological molecule to a functionalized chromophoricpolymer dot, the method comprising incubating a functionalizedchromophoric polymer dot with the biological molecule in a solutioncontaining a blocking agent under conditions suitable for conjugatingthe biological molecule to the functionalized chromophoric polymer dot,wherein the presence of the blocking agent in the solution reducesnon-specific adsorption of the biological molecule to the surface of thepolymer dot.

Generally, the blocking agent may be any molecule that reduces oreffectively eliminates non-specific binding of the target biologicalmolecule and polymer dot, therefore allowing proper conjugation toproceed. In one embodiment, the blocking agent is a water solublepolymers, for example, a polyalkylene glycol (e.g., a PEG), a PEO, apolypropylene glycol, a polyoxyalkylene, a starch, a poly-carbohydrate,a polysialic acid, and the like. In another embodiment, the blockingagent is a detergent, such as a non-ionic detergent or surfactant.Non-limiting examples of detergents that may be used include TritonX-100, Tween 20, Tween 80, a Brij detergent, and the like. In yetanother embodiment, the blocking agent is a carbohydrate, for example,dextran, amylose, glycogen, and the like.

Methods for Imaging and Molecular Labeling

As described above, the use of fluorescent polymers for in vivo imagingand molecular labeling has several advantages over the materialscurrently in use, including Qdots and doped latex particles. Forexample, fluorescent polymer dots possess high fluorescencebrightness/volume ratios, have high absorption cross sections, highradiative rates, high effective chromophore density, and minimal levelsof aggregation-induced fluorescence quenching. The use of fluorescentpolymer dots as fluorescent probes also confers other useful advantages,such as the lack of heavy metal ions that could leach out into solution.However, for applying these probes in biological imaging or sensingapplications several important problems have yet to be solved, inparticular, the surface chemistry and bioconjugation.

As described herein, the present invention provides numerous solutionsfor providing useful chromophoric polymer dot surface chemistry,functionalization and bio conjugation. Accordingly, the presentinvention provides improved methods for in vivo imaging and molecularlabeling comprising the use of functionalized chromophoric polymer dotsprovided herein.

In one embodiment, the present invention provides a method for labelinga target molecule in a biological sample, the method comprisingcontacting the biological sample with a bioconjugated chromophoricpolymer dot, wherein the bioconjugated chromophoric polymer dotcomprises a core and a cap, wherein said core comprises a chromophoricpolymer, and said cap comprises a functionalization agent attached to atargeting moiety via a reactive functional group, with the proviso thatnot all of the said cap is an organo silicate.

The targeting agent may be any molecule capable of specifically bindingto the target molecule of interest. In a preferred embodiment, thetargeting agent is an antibody or a fragment thereof. In another relatedembodiment, the targeting agent is an aptamer.

In a related embodiment, a method is provided for labeling a targetmolecule in a biological sample, the method comprising contacting thebiological sample with a bioconjugated chromophoric polymer dot, whereinthe bioconjugated chromophoric polymer dot comprises: (a) asemiconducting polymer; and (b) an amphiphilic molecule attached to atargeting moiety via a reactive functional group, wherein a portion ofthe amphiphilic molecule is embedded within the core of the polymer dotand the targeting moiety is located on the surface of the polymer dot.The targeting agent may be any molecule capable of specifically bindingto the target molecule of interest. In a preferred embodiment, thetargeting agent is an antibody or a fragment thereof. In another relatedembodiment, the targeting agent is an aptamer.

The targeted molecule of interest may be any molecule found inside anorganism, for example, a specific cell, protein, nucleic acid,carbohydrate, lipid, metabolite, and the like. In one specificembodiment, the target molecule is a molecule present on the surface ofa cell, for example a cancer cell. In a specific embodiment, thefunctional groups are selected from an alkyne, strained alkyne, azide,diene, alkene, cyclooctyne, and phosphine groups.

In another embodiment, the present invention provides a method for thebioorthogonal labeling of a cellular target, the method comprisingcontacting a cellular target having a first surface-exposed functionalgroup capable of participating in a click chemistry reaction with aclickable chromophoric polymer dot, wherein the clickable chromophoricpolymer dot comprises a core and a cap, wherein said core comprises achromophoric polymer, and said cap comprises a functionalization agentattached to a second functional group capable of reacting with the firstfunctional group in a click chemistry reaction, with the proviso thatnot all of the said cap is an organo silicate. In a specific embodiment,the functional groups are selected from an alkyne, strained alkyne,azide, diene, alkene, cyclooctyne, and phosphine groups.

In a related embodiment, the present invention provides a method for thebioorthogonal labeling of a cellular target, the method comprisingcontacting a cellular target having a first surface-exposed functionalgroup capable of participating in a click chemistry reaction with aclickable chromophoric polymer dot, wherein the clickable chromophoricpolymer dot comprises: (a) a semiconducting polymer; and (b) anamphiphilic molecule attached to a second functional group capable ofreacting with the first functional group in a click chemistry reaction,wherein a portion of the amphiphilic molecule is embedded within thecore of the polymer dot and the second functional group is located onthe surface of the polymer dot.

As described above, methods of incorporating a first functional groupcapable of participating in a click chemistry reaction into a moleculeof interest are known in the art. For example, an attractive approachfor installing azides into biomolecules is based on metabolic labeling,whereby an azide-containing biosynthetic precursor is incorporated intobiomolecules by using the cells' biosynthetic machinery.

Methods for the Detection of Cu²⁺ and Fe²⁺ Ions Using ChromophoricPolymer Dots

Copper and iron ions are two of the three most abundant transition metalions (including zinc) in the human body. The recommended intakes ofcopper and iron range from 0.8-0.9 mg/day and 8-18 mg/day for normaladults, respectively. The overdose of copper or iron, however, is knownto cause Cirrhosis of the liver, acute toxicity, acidosis, coagulopathy,and acute respiratory distress syndrome. According to the drinking waterstandards and health advisories revised by U.S. Environmental ProtectionAgency (EPA), the amount of copper and iron is limited to 1.0 mg/L and0.3 mg/L, respectively. As a result, there has been ongoing efforts todevelop better sensors for the detection of copper and iron, due totheir significance in the environment and in biological systems (Que E.L., et al., Chem. Rev., 2008, 108, 1517-1549). In the past decade,nanoparticle-based ion sensors have attracted intense interest becauseof their simplicity, good sensitivity and selectivity, high reliability,and relatively low cost. However, exploitation of semiconducting polymernanoparticles (Pdots) based fluorescence ion sensors remains unexplored.

Accordingly, in one aspect, the present invention provides a strategyfor the quantitative detection of copper and iron ion. This strategy isbased on fluorescence quenching induced by the aggregation of carboxylfunctionalized Pdots. A demonstration of this method can be found inExample 27.

In one aspect, the present invention provides a sensitive andratiometric approach for Cu²⁺ and Fe²⁺ ion detection based onchelation-mediated Pdot sensors. The linear detection range for bothCu²⁺ and Fe²⁺ fall within the physiologically relevant concentrationrange. However, this linear range can be further extended, if needed, bymodulating the PS-COOH density and/or the size of the Pdots. Thissimple, sensitive, and economical technique takes advantage of the highbrightness and optical tunability of semiconducting polymernanoparticles, and affords a means of rapid determination of Cu²⁺ andFe²⁺ for physiological or environmental analysis.

Accordingly, in one embodiment a method is provided for detecting copper(II) and/or iron (II) in a solution, the method comprising the steps of:(a) contacting a solution with a carboxyl functionalized semiconductingpolymer dot; and (b) detecting the level of fluorescence from thepolymer dot in the solution, wherein a reduction of fluorescence in thesolution, as compared to the fluorescence of the polymer dot in asolution not containing copper (II) or iron (II), is indicative that thesolution contains copper (II) and/or iron (II).

In one embodiment, the method further comprises the step of quantitatingthe level of copper (II) in the solution by: (c) adding a divalentcation chelating agent to the solution; (d) detecting the level offluorescence in the solution after the addition of the divalent cationchelating agent; and (e) determining the difference in the fluorescenceof the polymer dots in solution before and after the addition of thedivalent chelating agent, wherein the level of copper (II) in thesolution is determined by comparing the difference in the fluorescencewith a standard.

In another embodiment, the method further comprises the step ofquantitating the level of iron (II) in the solution by: (c) adding adivalent cation chelating agent to the solution; (d) detecting the levelof fluorescence in the solution after the addition of the divalentcation chelating agent; and (e) determining the difference between thefluorescence of the polymer dots in solution after the addition of thedivalent chelating agent and the fluorescence of the polymer dots in asolution not containing copper (II) or iron (II), wherein the level ofiron (II) in the solution is determined by comparing the difference inthe fluorescence with a standard.

EXAMPLES

The following examples are included to further describe the presentinvention, and should not be used to limit the scope of the invention.

A method for preparing the functionalized chromophoric polymer dot isdemonstrated, a process comprising the step of mixing a protic solventwith a mixture of chromophoric polymer and functionalization agent in anaprotic solution. The present invention also provides a bioconjugate,and its composition, which comprises the functionalized chromophoricpolymer dot and a biomolecule, wherein the biomolecule is attacheddirectly or indirectly to the functional group.

Example 1: Method for Preparing Functionalized Chromophoric Polymer Dots

The present example provides a method for obtaining a quantity offunctionalized chromophoric polymer dots for subsequent characterizationand biomolecular conjugation. FIG. 1 shows a schematic diagram forpreparing the functionalized chromophoric polymer dots and theirbiomolecular conjugates.

Functionalized chromophoric polymer dots in aqueous solution wereprepared as follows. First, a chromophoric polymer, for example PFBT,was dissolved in tetrahydrofuran (THF) by stirring under inertatmosphere to make a stock solution with a concentration of 1 mg/mL.Certain amount of functionalization agent, for example PS-PEG-COOH inTHF solution, was mixed with a diluted solution of PFBT to produce asolution mixture with a PFBT concentration of 40 μg/mL and a PS-PEG-COOHconcentration of 4 μg/mL. The mixture was stirred to form homogeneoussolutions. A 5 mL quantity of the solution mixture was added quickly to10 mL of deionized water while sonicating the mixture. The THF wasremoved by nitrogen stripping, and the solution was concentrated bycontinuous nitrogen stripping to 4 mL on a 90° C. hotplate, followed byfiltration through a 0.2 micron filter. The resulting nanoparticledispersions are clear and stable for months with no signs ofaggregation.

Example 2: AFM Characterization of Functionalized Chromophoric PolymerDots

The functionalized chromophoric polymer dots prepared according to themethod provided in Example 1 were assessed by AFM for their size,morphology and monodispersity. For the AFM measurements, one drop of thenanoparticle dispersion was placed on freshly cleaved mica substrate.After evaporation of the water, the surface was scanned with a DigitalInstruments multimode AFM in tapping mode. FIG. 2A shows arepresentative AFM image of the functionalized chromophoric polymerdots. A particle height histogram taken from the AFM image indicatesthat most particles possess diameters in the range of 10-20 nm (FIG.2B). The lateral dimensions are also in the range of 10-20 nm after thetip width is taken into account. The morphology and size are consistentwith those of the polymer dots prepared without functionalizationpolymer, indicating the presence of a small amount of amphiphilicpolymer has no apparent effect on particle size and morphology. Inaccordance with the preparation method in Example 1, the functionalizedchromophoric polymer dots can be prepared with their size ranging from 2nm to 1000 nm by adjusting the injection concentration of thechromophoric polymer.

Example 3: Optical Characterization of Functionalized ChromophoricPolymer Dots

The functionalized chromophoric polymer dots prepared according to themethod provided in Example 1 were assessed for their fluorescenceproperties. UV-Vis absorption spectra were recorded with a DU 720spectrophotometer using 1 cm quartz cuvettes. Fluorescence spectra werecollected with a Fluorolog-3 fluorometer using a 1 cm quartz cuvette.The functionalized chromophoric polymer dots exhibit similar absorptionand emission spectra to those of bare chromophoric polymer dots (FIG.3), indicating the surface functionalization does not effect the opticalproperties of the polymer dots. Depending on the chromophoric polymerspecies, the chromophoric polymer dots exhibit absorption bands rangingfrom 350 nm to 550 nm, a wavelength range that is convenient forfluorescence microscopy and laser excitation. FIG. 3 shows theabsorption and emission spectra of functionalized chromophoric polymerPFBT. Analysis of the UV-Vis absorption spectra at a known particleconcentration indicated that the peak absorption cross section of singleparticles (˜15 nm diameter) were about 2×10-13 cm², roughly ten to onehundred times larger than that of CdSe quantum dots in the visible andnear-UV range, and roughly three orders of magnitude larger than typicalorganic fluorescent dyes.

Fluorescence quantum yield of the PFBT dots was determined to be 30%using a diluted solution of Coumarin 6 in ethanol as standard. Thefluorescence brightness is defined as the product of absorption crosssection and quantum yield results. To the best of our knowledge, thefluorescence brightness of the chromophoric polymer dots exceeds that ofany other nanoparticle of the same size under typical conditions. Thesize of the particle does not appear to have an appreciable effect onthe shape of the absorption and fluorescence spectra—the principaleffect of increased particle size is an increase in the absorptioncross-section and brightness. This property facilitates adjustment ofparticle size and brightness to meet the demands of a particularapplication, and is in contrast with colloidal semiconductor quantumdots.

Example 4: Conjugation of Biomolecules to Functionalized ChromophoricPolymer Dots

This example demonstrates that functionalized chromophoric polymer dotscan be conjugated to biomolecules for subsequent imaging of cellularstructure, or any other fluorescence based biological detection. Thefunctionalized chromophoric polymer dots prepared in accordance with themethod of the present invention can be conjugated to any biomolecules,such as protein and antibodies, which contain primary amino functionalgroups. Carboxyl groups can be reacted to N-hydroxysuccinimide (NHS) inthe presence of a carbodiimide such as EDC to produce amine-reactiveesters of carboxylate groups for crosslinking with primary amine groups.In a typical bioconjugation reaction, 20 μL of EDC (5 mg/mL in MilliQwater) and 10 μL of NHS (5 mg/mL in MilliQ water) were added to 1 mL offunctionalized chromophoric polymer dots (40 μg/mL in MilliQ water). Theabove mixture was left on a rotary shaker for 30 minutes for activation.Then 20 μL of polyethylene glycol (5% w/v PEG) and 20 μL of concentratedHEPES buffer (1 M) were added, resulting in a solution of the activatedpolymer dots in 20 mM HEPES buffer with a pH of 7.3. Finally, 40 μL ofStreptavidin or IgG antibody (1 mg/mL) was added to the solution, andthe reaction last for 4 hours at room temperature. The resultingchromophoric polymer dot bioconjugates were separated from freebiomolecules by gel filtration using Sephacryl HR-300 as the media.

Example 5: Use of Chromophoric Polymer Dot-IgG Conjugates for CancerMarker Detection in Live Cells

This example provides a demonstration using the chromophoric polymer dotbioconjugates to detect cancer markers in human breast cancer cells. Thebreast cancer cell line SK-BR-3 was cultured in McCoy's 5A mediumsupplemented with 10% Fetal Bovine Serum and 1% penicillin/streptamycin.A million cells were harvested from the culture flask, washed, andresuspended in 100 μL labeling buffer (1×PBS, 2 mM EDTA, 0.5% BSA). Thecell suspension was incubated with primary anti-human CD326 (EpCAM)antibody on a shaker for 30 minutes at dark and room temperature,followed by two washing steps using labeling buffer. Then the cells wereincubated with the chromophoric polymer dot secondary IgG conjugates for30 min on a shaker at dark and room temperature, followed by another twowashing steps. A drop of cell suspension was placed on a coverslip,covered with a glass slide, and imaged immediately under a fluorescenceconfocal microscope (Zeiss LSM 510).

As shown in FIG. 4A, the chromophoric polymer dot-IgG probe successfullylabeled EpCAM receptors on the surface of human SK-BR-3 breast cancercells after the cells were incubated with a primary anti-EpCAM antibody.When the primary antibody is absent, i.e., the cells were incubated withthe polymer dot-IgG alone, little or no signal was detected (FIG. 4B),indicating that the polymer dot-IgG conjugates are specific for thetarget.

Example 6: Use of Chromophoric Polymer Dot-Streptavidin Conjugates forSubcellular Imaging

This example provides a demonstration using the chromophoric polymer dotbioconjugates to detect subcellular structures. The breast cancer cellline MCF-7 was cultured in Eagles minimum essential medium supplementedwith 10% Fetal Bovine Serum and 1% penicillin/streptomycin. Ten thousandcells were plated on a 22×22 mm glass coverslip, cultured using theabove medium in a 6-well plates until the density reach 50-70%confluence. The cells were fixed with 4% paraformaldehyde for 15minutes, permeabilized with 0.25% Triton-X 100 in PBS for 15 minutes,and blocked in 2% BSA (w/v) for 30 minutes. To label microtubules, thefixed and BSA-blocked MCF-7 cells were incubated sequentially withbiotinylated monoclonal anti-α-tubulin antibody for one hour, andchromophoric polymer dot-streptavidin conjugates for 30 minutes. Thecoverslip with the stained cells were mounted on a glass slide andimaged with the fluorescence confocal microscope (Zeiss LSM 510). Asshown in FIG. 5A, microtubules were clearly labeled with thechromophoric polymer dot-streptavidin. When the cells were incubatedwith the chromophoric polymer dot-streptavidin alone, very weak or noapparent signal was detected (FIG. 5B), indicating that the polymerdot-streptavidin conjugates are specific for the labeling.

Example 7: Functionalization of Semiconducting Polymer Dots

Fluorescent semiconducting polymerPoly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}thiadiazole)](PFBT, MW 157,000, polydispersity 3.0) was purchased from ADS Dyes, Inc.(Quebec, Canada). A comb-like polymer, polystyrene grafted with ethyleneoxide functionalized with carboxyl groups (PS-PEG-COOH, main chain MW8,500, graft chain MW 1,200, total chain MW 21,700, polydispersity1.25), was purchased from Polymer Source Inc. (Quebec, Canada). Allother reagents were purchased from Sigma-Aldrich (St. Louis, Mo., USA),and all experiments were performed at room temperature unless indicatedotherwise.

Functionalized Pdots in aqueous solution are prepared by using amodified nano-precipitation method. First, PFBT was dissolved intetrahydrofuran (THF) to make a stock solution with a concentration of 1mg/mL. PS-PEG-COOH was also dissolved in THF and mixed with a dilutedsolution of PFBT to produce a solution mixture with a PFBT concentrationof 50 μg/mL and a PS-PEG-COOH concentration ranging from 0 to 10 μg/mL.The mixture was sonicated to form homogeneous solutions. A 5 mL quantityof the solution mixture was added quickly to 10 mL of MilliQ water in abath sonicator. The THF was removed by nitrogen stripping, and thesolution was concentrated by continuous nitrogen stripping to 5 mL on a90° C. hotplate, followed by filtration through a 0.2 micron filter. Theresulting functionalized Pdot dispersions are clear and stable formonths without signs of aggregation.

Example 8: Physical Characterization of Functionalized SemiconductingPolymer Dots

Functionalized PFBT dots were prepared using a precursor solutionmixture with a constant PFBT concentration and PS-PEG-COOH/PFBTfractions ranging from 0 to 20 weight percent. The size and morphologyof the functionalized PFBT dots were characterized by atomic forcemicroscopy (AFM, FIG. 7A). Particle height histogram obtained from AFMimages indicated that the majority of PFBT dots possessed diameters inthe range of 10±3 nm (FIG. 7B). In comparison with the unfunctionalizedPdots, the presence of a small amount of PS-PEG-COOH polymer (<20 wt %)did not cause any noticeable effects on particle size and morphology.Absorption and emission spectra of Pdots do not change with size (Wu,C.; Bull, B.; Szymanski, C.; Christensen, K.; McNeill, J. ACS Nano 2008,2, 2415-2423), and thus this size-independent feature of Pdotsignificantly relaxes the constraint on size control in nanoparticlepreparation. Moreover, this size-independence feature may beadvantageous to obtain brighter probes for certain applications becausea larger size merely increases the brightness of the probe. It should benoted that this functionalization strategy led to effective nanoparticleprobes in terms of fluorophore density; for example, more than 80percent of the semiconducting polymer nanoparticles can be effectivefluorophores. In contrast, for Qdots and dye-loaded spheres, theeffective fluorophores are limited to a few percent of the particlevolume or weight due to the presence of a thick encapsulation layer (forQdots) or self-quenching of dyes (for dye-doped spheres).

Example 9: Optical Characterization of Bioconjugated SemiconductingPolymer Dots

The cell-labeling brightness of Pdot bioconjugates with those ofcommercially available Qdot 565-streptavidin and Alexa-IgG probes wasfirst quantified using a microfluidic flow cytometer. The flow-throughexperiments were conducted with a microfluidic chip with 200 μm wide and50 μm high straight channels on an inverted light microscope equippedwith a 20×NA 0.4 objective (Nikon Eclipse TE 2000-U, Melville, N.Y.,USA). The as-labeled cell suspensions (10,000 per ml) were introducedinto the rectangular channel using a syringe pump at 50 μl min-1 for upto 5 minutes. A 488 nm sapphire laser (Coherent, Santa Clara, Calif.)was guided into the microscope to excite the sample. Before each sampleacquisition, the laser power was measured in the path of light beforeentering the microscope using a power meter. Fluorescence signal wasfiltered by a 500 nm long pass filter (HQ500LP; Chroma, Rockingham, Vt.,USA), and collected by a Single Photon Counting Module (APD, PerkinElmerSPCM-QC9-QTY2, Salem, Mass., USA). A personal computer and a LabViewcoded program (National Instruments Corporation, Austin, Tex., USA) wereused to read out the signals of the SPCM at a sampling frequency of 10kHz. The raw APD counts for each sample were stored in text files andconverted in to frequency plots using a custom-coded Maple 5.1 program(MapleSoft, Waterloo, ON, Canada).

The cell-labeling brightness of Pdot-streptavidin and Qdot565-streptavidin probes was also quantified by analyzing fluorescenceimages of the labeled MCF-7 cells. A drop of cell suspension was placedon a coverslip, covered with a glass slide, and viewed on an uprightmicroscope with an AZ-Plan Apo 4×NA 0.4 objective (Nikon AZ100,Melville, N.Y., USA). The excitation light was provided with a fiberilluminator (130 W mercury lamp), and filtered by a band pass filter(Semrock FF01-482/35-25, Rochester, N.Y. USA). Fluorescence signal wasfiltered by a 520 nm long pass filter (HQ520LP; Chroma, Rockingham, Vt.,USA), and imaged on a CCD camera (Prosilica GC1380, Newburyport, Mass.).Fluorescence images were processed with a custom-coded Labview program,and intensity distributions of single-cell labeling brightness wereobtained (FIG. 12).

Example 10: Single Particle Imaging of PFBT Pdots

A useful estimate of fluorescence brightness is given by the product ofthe peak absorption cross section and the fluorescence quantum yield.Photophysical data indicate that PFBT dots of ˜10 nm diameter are about30 times brighter than IgG-Alexa 488, and Qdot 565 probes under atypical laser excitation (488 nm). A side-by-side brightness comparisonwould provide further evidence of the extraordinary brightness of Pdots.We carried out single-particle imaging to experimentally evaluate andcompare the brightness and photostability of the three probes.

Briefly, fluorescent samples were diluted in Milli-Q water, dried undervacuum on cleaned glass coverslips, and imaged on a fluorescencemicroscope. The 488-nm laser beam from a sapphire laser (Coherent, SantaClara, Calif. USA) was directed into an inverted microscope (NikonTE2000U, Melville, N.Y., USA) using lab-built steering optics. Laserexcitation power was measured at the nosepiece before the objective. Theobjective used for illumination and light collection was a 1.45 NA60×TIRF objective (Nikon, Melville, N.Y., USA). Fluorescence signal wasfiltered by a 500 nm long pass filter (HQ500LP; Chroma, Rockingham, Vt.,USA) and imaged on an EMCCD camera (Photometrics Cascade: 512B, Tucson,Ariz. USA). Because saturation of the detector was observed for somePdot particles in FIG. 8, a neutral density filter (optical density of1.5) was placed together with the emission filter when imaging Pdotsamples. Fluorescence intensity of Pdot particles was back-calculatedaccording to the attenuation factor. Single-particle photobleachingmeasurements were obtained by acquiring a series of consecutive frames.Fluorescence intensity emitted per frame for a given particle wasestimated by integrating the CCD signal over the fluorescence spot.

FIGS. 8A, 8B, and 8C show typical single-particle epi-fluorescenceimages of PFBT dots, IgG-Alexa 488, and Qdot 565, respectively, obtainedunder identical acquisition and laser excitation conditions. With arelative low excitation power (1 mW) from a 488 nm laser, very bright,near-diffraction-limited spots were clearly observed for individual PFBTdots. Some Pdots actually saturated the detector (FIG. 8A), whereas theIgG-Alexa 488 and Qdots exhibited much lower intensity levels, barelydetected by the camera at the low excitation power we used (FIG. 8B,8C). The PFBT dots exhibited an order-of-magnitude improvement insignal-to-background ratio compared to those of Qdot 565 and IgG-Alexa488 (FIG. 8D). Such a prominent contrast is primarily due to the highper-particle absorption cross section of Pdots, which would beparticularly suitable for fluorescence detection requiring lowexcitation conditions. For further comparing the probe performance, weincreased laser excitation power to 4 mW so that Qdot 565 and IgG-488probes can be sufficiently detected by the camera. Because saturation ofthe detector was observed for Pdot particles, a neutral density filter(optical density of 1.5, which blocks 97% of the emitted fluorescence)was placed together with the emission filter when imaging Pdot samples,and their fluorescence intensities were back-calculated according to theattenuation factor. For all the three probes, background was subtracted.Fluorescence intensity distribution of several thousands particlesindicated that PFBT dots were ˜30 times brighter than IgG-Alexa 488 andQdot 565 (FIG. 8E), consistent with the brightness comparison based onthe photophysical parameters.

Single-particle photobleaching measurements indicated excellentphotostability of PFBT dots (FIG. 8F). Statistical analyses of multiplephotobleaching trajectories showed that over 10⁹ photons per Pdot wereemitted prior to photobleaching, two or three orders of magnitude largerthan those emitted by individual Qdot 565 and IgG-Alexa 488 particles.Furthermore, a large number of photons could be obtained from individualPdots at high acquisition rates (200,000 photons detected per Pdot per20 ms exposure) because of their high brightness, short fluorescencelifetime, and the presence of multiple emitters per particle. Thisfeature was recently exploited to yield a particle tracking uncertaintyof ˜1 nm (Yu, J.; Wu, C.; Sahu, S.; Fernando, L.; Szymanski, C.;McNeill, J. J. Am. Chem. Soc. 2009, 131, 18410-18414), which makes Pdotsfar superior in high-speed single-particle tracking experiments thanconventional fluorescent dyes and Qdots. It is worth noting that mostPFBT dots exhibit continuous emission behavior without any obviousfluorescence blinking while most Qdots exhibit pronounced blinking (FIG.8F). This non-blinking feature of Pdots is particularly valuable insingle-molecule applications.

Example 11: Elimination of Non-Specific Binding to the Surface ofFunctionalized Pdots

In a first attempt to functionalize Pdots, streptavidin was selectedbecause most biological labeling molecules, such as antibodies, can beeasily derivatized with biotin. However, because the relatively largesurface area of Pdots is intrinsically hydrophobic, although surfacemodification tends to make it more hydrophilic, there is a concern thatbiomolecules will be non-specifically adsorbed onto the Pdot surface.This concern was verified for carboxyl functionalized Pdots. Briefly,carboxyl functionalized Pdots were mixed with and without streptavidinin a buffered solution, in the absence of a coupling reagent that linkscarboxyl groups to amine groups on biological molecules, and thenincubated with biotin silica beads. After centrifugation, the Pdots thathad been incubated with streptavidin were clearly retained in the pelletof the biotin silica beads, and those incubated without streptavidinshowed no binding to the beads (FIG. 7C), thus indicating severenon-specific adsorption of streptavidin onto the Pdot surface.

To overcome this non-specific adsorption, Pdots were mixed withstreptavidin in a buffer solution containing 0.1 wt % polyethyleneglycol (PEG). The resulting Pdots showed no detectable binding to biotinsilica beads, suggesting that the presence of PEG significantly reducednon-specific adsorption (FIG. 7C). Accordingly, covalent bioconjugationwas successfully performed in a PEG-containing buffer. The peptide bondformation between the carboxyl groups on Pdots and the amine groups ofstreptavidin was catalyzed by a carbodiimide such as1-ethyl-3[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC). TheEDC-catalyzed, Pdot-streptavidin conjugates showed clear binding tobiotin silica beads; while binding was not observed for the productsobtained in the absence of EDC (FIG. 7C). In a separate control,identical bioconjugation conditions (i.e. streptavidin and EDC in PEGcontaining buffer) were employed with bare, non-functionalized Pdots.Binding was not detectable on biotin beads, further confirming that thebioconjugation of streptavidin to Pdots was covalent and that labelingof strepavidin-Pdots to biotin beads was specific and without anydetectable non-specific binding.

The Pdots may be further passivated with additives such as bovine serumalbumin (BSA), which can maintain long-term colloidal stability, blockhydrophobic surfaces, and reduce non-specific binding in labelingexperiments. We found BSA-passivated Pdot bioconjugates are stable formonths at physiological pH in HEPES, PBS, Tris, and borate buffers. FIG.7D inset shows two photographs of PFBT-streptavidin conjugates in 1×PBSbuffer after 6 months of storage. The suspension of PFBT conjugates wasstable, clear (not turbid), and exhibited strong fluorescence under UVlamp illumination (365 nm).

Example 12: Biomolecular Conjugation to Functionalized Pdots

Streptavidin and goat anti-mouse IgG antibodies were purchased fromInvitrogen (Eugene, Oreg., USA). Bionconjugation was performed byutilizing the EDC-catalyzed reaction between carboxyl groups on Pdotsand amine groups on biomolecules. In a typical bioconjugation reaction,20 μL of polyethylene glycol (5% w/v PEG, MW 3350) and 20 μL ofconcentrated HEPES buffer (1 M) were added to 1 mL of functionalizedPdot solution (50 μg/mL in MilliQ water), resulting in a Pdot solutionin 20 mM HEPES buffer with a pH of 7.3. Then, 40 μL of streptavidin orIgG antibody (1 mg/mL) was added to the solution and mixed well on avortex. Last, 20 μL of freshly-prepared1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)solution (5 mg/mL in MilliQ water) was added to the solution, and theabove mixture was left on a rotary shaker for 4 hours at roomtemperature. Finally, the resulting Pdot bioconjugates were separatedfrom free biomolecules by gel filtration using Sephacryl HR-300 gelmedia.

Example 13: Cell Culture

The breast cancer cell lines MCF-7 and SK-BR-3 were ordered fromAmerican Type Culture Collection (ATCC, Manassas, Va., USA). Cells werecultured at 37° C., 5% CO₂ in Eagles minimum essential medium (forMCF-7) or McCoy's 5A medium (for SK-BR-3) supplemented with 10% FetalBovine Serum (FBS), 50 U/mL penicillin, and 50 μg/mL streptomycin. Thecells were pre-cultured prior to experiments until confluence wasreached. The cells were harvested from the culture flask by brieflyrinsing with culture media followed by incubation with 5 mL ofTrypsin-EDTA solution (0.25 w/v % Trypsin, 0.53 mM EDTA) at 37° C. for5-15 minutes. After complete detachment, the cells were rinsed,centrifuged, and resuspended in labeling buffer (1×PBS, 2 mM EDTA, 1%BSA). The cell concentration was determined by microscopy using ahemacytometer.

Example 14: Use of Chromophoric Polymer Dot-IgG Conjugates for Detectionof CD326 and CD340 Cell Markers

It was previously demonstrated that bare Pdots could be delivered intocultured cells, presumably by endocytosis. However, when Pdotsnon-specifically bound to the cell surface; specific cellular targetswere not labeled (see, for example, Wu, C.; Bull, B.; Szymanski, C.;Christensen, K.; McNeill, J. ACS Nano 2008, 2, 2415-2423; Pu, K. Y.; Li,K.; Shi, J. B.; Liu, B. Chem. Mater. 2009, 21, 3816-3822; Howes, P.;Green, M.; Levitt, J.; Suhling, K.; Hughes, M. J. Am. Chem. Soc. 2010,132, 3989-3996; and Rahim, N. A. A.; McDaniel, W.; Bardon, K.;Srinivasan, S.; Vickerman, V.; So, P. T. C.; Moon, J. H. Adv. Mater.2009, 21, 3492-3496). Therefore, it was unclear from these studieswhether Pdot probes could be made specific enough to recognize cellulartargets for effective labeling in real applications.

For a cell labeling experiment, Qdot 565-streptavidin, Alexa 488-IgG,and BlockAid™ blocking buffer were purchased from Invitrogen (Eugene,Oreg., USA). Pdot bioconjugates were synthesized using the methods asdescribed above.

For labeling a cell-surface marker with IgG conjugates, a million cellsin 100 μL labeling buffer was incubated with 5 μg/mL primary anti-humanCD326 antibody (anti-EpCAM, Biolegend, San Diego, Calif., USA) for MCF-7cells, or 5 μg/mL primary anti-human CD340 (anti-Her2, Biolegend, SanDiego, Calif., USA) on a rotary shaker for 30 minutes in the dark and atroom temperature, followed by a washing step using labeling buffer. Thenthe cells were incubated with 5 nM Pdot-IgG or Alexa 488-IgG conjugatesfor 30 minutes on a shaker in the dark and at room temperature, followedby another two washing steps. For labeling cell-surface marker withstreptavidin conjugates, a million MCF-7 cells in 100 μL labeling bufferwas incubated sequentially with 5 μg/mL primary anti-human CD326antibody, 5 μg/mL biotinylated secondary anti-mouse IgG (Biolegend, SanDiego, Calif., USA), and 5 nM Pdot-streptavidin or Qdot 565-steptavidin(Invitrogen, Eugene, Oreg., USA) for 30 minutes each, followed byanother two washing steps. A drop of cell suspension was placed on acoverslip, covered with a glass slide, and imaged immediately under afluorescence confocal microscope (Zeiss LSM 510).

Streptavidin and IgGs are widely used in bioconjugation forimmunofluorescent labeling of cellular targets. We created Pdot-IgG andPdot-streptavidin probes and investigated their ability to label aspecific cellular target, EpCAM/CD326, an epithelial cell-surface markercurrently used for the detection of circulating tumor cells. FIG. 9Ashows the Pdot-IgG probes successfully labeled EpCAM receptors on thesurface of live MCF-7 human breast cancer cells after the cells wereincubated with a monoclonal primary anti-EpCAM antibody. When the cellswere incubated with just the Pdot-IgG alone, in the absence of theprimary antibody, cell-labeling was not detected (FIG. 9A, bottom),indicating that the Pdot-IgG conjugates are highly specific for thetarget.

Next, Pdot-streptavidin conjugates were used as an alternative probe todetect EpCAM. The Pdot-streptavidin probes, together with the primaryanti-EpCAM antibody and biotinylated goat anti-mouse IgG secondaryantibody, also effectively labeled EpCAM on the surface of live MCF-7cells (FIG. 9B). When the cells were incubated with primary antibody andPdot-streptavidin in the absence of biotin anti-mouse IgG, nofluorescence was observed on the cell surface (FIG. 9B, bottom), thusagain demonstrating the highly specific binding of Pdot-strepavidin. Thelack of signal also indicated the absence of nonspecific binding in thisbiotin-streptavidin labeling system.

Pdot bioconjugates were then used to label another cell-surface marker,Her2 (target of the anti breast cancer drug, Heceptin), on a differentcell line SK-BR-3, as well as subcellular structures such asmicrotubules in fixed MCF-7 cells (FIG. 5). Pdot bioconjugates in bothcases labeled the targets specifically and effectively, demonstratingtheir comprehensive application to cell labeling.

Example 15: Use of Chromophoric Polymer Dot-IgG Conjugates for theLabeling of Microtubules

For microtubule labeling, ten thousands of MCF-7 cells were plated on a22×22 mm glass coverslip, cultured until the density reach 60-70%confluence. The cells were fixed with 4% paraformaldehyde for 15minutes, permeabilized with 0.25% Triton-X 100 in PBS for 15 minutes,and blocked in 2% BSA (w/v) for 30 minutes. The fixed and BSA-blockedMCF-7 cells were incubated sequentially with 5 μg/mL biotinylatedmonoclonal anti-α-tubulin antibody (Biolegend, San Diego, Calif., USA)for 60 minutes, and 10 nM Pdot-streptavidin conjugates for 30 minutes.The stained cells were mounted on a glass slide and imaged with thefluorescence confocal microscope (Zeiss LSM 510).

Example 16: Use of Chromophoric Polymer Dot Conjugates in Flow Cytometry

Besides fluorescence imaging, flow cytometry is another area where thebrightness of probes is important. The labeling brightness of Pdotbioconjugates was compared to that of commercially availableQdot-streptavidin and Alexa-IgG probes using a microfluidic flowcytometer. FIG. 10A shows the flow-through detection of MCF-7 cellslabeled with Pdot-streptavidin. At the lowest excitation intensity used(0.1 mW), a well-defined intensity peak for the Pdot-labeled cellsappeared far above the background. In contrast, the peak forQdot-labeled cells was not clearly separated from the background (FIG.10B). The Pdot peak moved to higher intensity with increasing excitationintensity and started to saturate the detector at a laser power of 0.5mW. In all excitation conditions, MCF-7 cells labeled withPdot-streptavidin exhibited much higher intensity levels compared to theresults of Qdot-labeled cells using the same labeling concentration asPdot-streptavidin. The Pdot probes could provide significantly highersignal level at low excitation conditions, a very useful benefit forbiological detection in optically turbid media such as blood or thicktissues.

Similar intensity comparisons were performed using Pdot-IgG and Alexa488-IgG probes using the microfluidic flow cytometer (FIG. 11).Quantitative analyses of the flow cytometry data showed that the averageintensity of Pdot-labeled cells is ˜25 times brighter than theQdot-labeled ones (FIG. 10C), and ˜18 times brighter than Alexa-IgGlabeled cells (FIG. 10D). We further quantified the labeling brightnessby analyzing fluorescence images of MCF-7 cells labeled with eitherPdot-streptavidin or Qdot-streptavidin. The Pdot-labeled cells were ˜20times brighter than the Qdot-labeled ones, consistent with the flowcytometry data (FIG. 12).

These cell-labeling comparison values are slightly lower than thoseobtained from single-particle imaging. The lower values may beattributed to several factors, such as discrepancies in collectiveemission behavior of probe assemblies compared to individual particles;change in binding constants of antibody or streptavidin uponbioconjugation; or variation in emission rate with excitation intensity(saturation). It is also worth noting that cell labeling was performedaccording to the optimized concentrations for Qdot-streptavidin andAlexa-IgG probes which may not be optimal for Pdot probes. Therefore,the present comparison is a conservative estimate of the advantagesprovided by Pdot bioconjugates over traditional dye and Qdotbioconjugates. More detailed work is needed for optimizing thebioconjugation reactions, as well as the labeling conditions, for thisnew class of Pdot-based probes. Nevertheless, the current cell imagingand flow cytometry results clearly indicate that Pdot labeling providessignificant improvements in signal level compared to commerciallyavailable Alexa-IgG and Qdot probes.

Example 17: Preparation and Azido- and Alkyne-Pdots Compatible withClick Chemistry Applications

The fluorescent semiconducting polymerpoly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}-thiadiazole)](PFBT, MW 157,000, polydispersity 3.0) was purchased from ADS Dyes, Inc.(Quebec, Canada). The copolymer poly(styrene-co-maleic anhydride) (PSMA,cumene terminated, average Mw˜1,700, styrene content 68%) was purchasedfrom Sigma-Aldrich (St. Louis, Mo., USA)). All other reagents fornanoparticle preparation were purchased from Sigma-Aldrich (St. Louis,Mo., USA).

Functionalized Pdots in aqueous solution were prepared by using amodified nano-precipitation method. All experiments were performed atroom temperature unless indicated otherwise. In a typical preparation,the fluorescent semiconducting polymer PFBT was first dissolved intetrahydrofuran (THF) to make a 1 mg/mL stock solution. The copolymerPSMA was also dissolved in THF and mixed with a diluted solution of PFBTto produce a solution mixture with a PFBT concentration of 50 μg/mL anda PSMA concentration of 20 μg/mL. The mixture was sonicated to form ahomogeneous solution. A 5 mL quantity of the solution mixture wasquickly added to 10 mL of MilliQ water in a bath sonicator. The THF wasremoved by nitrogen stripping. The solution was concentrated bycontinuous nitrogen stripping to 5 mL on a 90° C. hotplate followed byfiltration through a 0.2 micron filter. During nanoparticle formation,the maleic anhydride units of PSMA molecules were hydrolyzed in theaqueous environment, generating carboxyl groups on Pdots. The Pdotdispersions were clear and stable for months without signs ofaggregation.

Surface conjugation was performed by utilizing the EDC-catalyzedreaction between carboxyl Pdots and the respective amine-containingmolecules. 11-Azido-3,6,9-trioxaundecan-1-amine was used to formazido-Pdots. Propargylamine was used to produce alkyne-Pdots.Amine-terminated poly(ethylene glycol) was used to form PEG-Pdots. In atypical conjugation reaction, 60 μL of polyethylene glycol (5% w/v PEG,MW 3350) and 60 μL of concentrated HEPES buffer (1 M) were added to 3 mLof carboxyl Pdot solution (50 μg/mL in MilliQ water), resulting in aPdot solution in 20 mM HEPES buffer with a pH of 7.3. Then, 30 μL ofamine-containing molecules (1 mg/mL) was added to the solution and mixedwell on a vortex. Last, 60 μL of freshly-prepared EDC solution (5 mg/mLin MilliQ water) was added to the solution, and the above mixture wasmagnetically stirred for 4 hours at room temperature. Finally, theresulting Pdot conjugates were separated from free molecules by Bio-RadEcono-Pac® 10DG columns (Hercules, Calif., USA).

Example 18: Characterization of Clickable Pdots

Fluorescence spectra were obtained using a commercial Fluorolog-3fluorometer (HORIBA Jobin Yvon, NJ USA). Analysis of the absorptionspectrum for ˜15 nm-diameter PFBT dots indicated a peak extinctioncoefficient of 5.0×10⁷ M⁻¹cm⁻¹ (FIG. 15). Fluorescence quantum yield ofthe functionalized Pdots was determined to be 0.28 using a dilutesolution of Coumarin 6 in ethanol as standard. The large extinctioncoefficient and high quantum yield indicate much higher per-particlebrightness as compared to other fluorescent nanoparticles.Single-particle photobleaching studies of the functionalized Pdotsshowed that over 10⁹ photons per Pdot were emitted prior tophotobleaching, consistent with their excellent photostability (C. Wu,B. Bull, C. Szymanski, K. Christensen, J. McNeill, ACS Nano 2008, 2,2415).

Gel electrophoresis was performed to characterize the formation ofdifferent functional groups on the Pdot surface using a 0.7% agarose gel(FIG. 17b ). Compared with unfunctionalized, bare Pdots, thecarboxyl-functionalized Pdots exhibited an apparent increase in mobilityin the gel. Briefly, agarose gel electrophoresis of the functionalizedPdots was carried out using a Mupid®-exU submarine electrophoresissystem. Pdots (in 30% glycerol) were loaded onto a 0.7% agarose gelcontaining 0.1% PEG. The Pdot-loaded gel was run for 20 min at 135 V intris-borate-EDTA (TBE) buffer, and then imaged on Kodak image station440CF system.

Dynamic light scattering and transmission electron microscopy (TEM)measurements showed that both the bare and the functionalized Pdots hadcomparable particle sizes, with an average of ˜15 nm in diameter (FIG.17c ). Therefore, the high mobility of PSMA-functionalized Pdotsindicated the formation of negatively charged carboxyl groups on thePdot surface. For the TEM measurements, one drop of the Pdot dispersionwas placed on a carbon-coated copper grid. After evaporation of thewater, the nanoparticles were imaged with a transmission electronmicroscope (FEI Tecnai F20). UV-Vis absorption spectra were recordedwith a DU 720 scanning spectrophotometer (Beckman Coulter, Inc., CA USA)using 1 cm quartz cuvettes.

Surface conjugation was performed with different amine-containingmolecules (amine-terminated polyethylene glycol (PEG), azide, andalkyne). Dynamic light scattering of the conjugated Pdots showed noobvious change in particle size because the conjugation was with smallmolecules. However, they exhibited shifted migration bands in the gel asanticipated, due to the reduced charges of the Pdot conjugates comparedto the carboxyl-functionalized Pdots. These results clearly indicatesuccessful carboxyl functionalization of the Pdots as well as all thesubsequent surface modifications.

Example 19: Stability of Functionalized Pdots

The pH and ion sensitivity of Pdot fluorescence in biologicalapplications was analyzed, particularly the copper-catalyzed clickchemistry. It was found that the fluorescence of Pdots was not affectedby most biologically relevant ions, including iron, zinc, and copper,three of the most abundant ions in biological organisms. The Pdotfluorescence is also independent of pH in the range of 4 to 9 (FIG. 16).This fact can be attributed to the hydrophobic organic nature of Pdots,which tend not to have any chemical interaction with ionic species. Incontrast, inorganic Qdots are significantly quenched by copper and ironions (H. Y. Xie, H. G. Liang, Z. L. Zhang, Y. Liu, Z. K. He, D. W. Pang,Spectrochimica Acta Part A 2004, 60, 2527). As shown in FIG. 17a , PFBTdots remained highly fluorescent in MilliQ water containing a high Cu⁺concentration of 1 mM, whereas Qdots were completely quenched at a muchlower Cu⁺ concentration of 1 μM (S. Han, N. K. Devaraj, J. Lee, S. A.Hilderbrand, R. Weissleder, M. G. Bawendi, J. Am. Chem. Soc. 2010, 132,7838). This property provides a significant advantage for applying Pdotsin various studies based on copper (I)-catalyzed click reactions. Toprevent self aggregation of Pdots in the copper solution, we added PEGinto the solution.

Example 20: Reactivity of Clickable Pdots

FIG. 17d shows a fluorescence assay examining the reactivity ofazido-Pdots towards a terminal alkyne group via copper (I)-catalyzedclick reaction. When mixed with a copper solution, the azido-Pdotsexhibited an emission intensity similar to that of the pure Pdots,confirming that their fluorescence is insensitive to copper ions. Aslight decrease in intensity was observed in the mixture of Pdots andalkyne-Alexa 594 (no Cu (I)), but this was primarily due to the innerfilter effect rather than direct quenching caused by fluorescenceresonance energy transfer (FRET). In contrast, when directly linked toalkyne-Alexa 594 in the presence of Cu (I), the azido-Pdots showedremarkable fluorescence quenching accompanied by an emission peak fromthe Alexa dye. This spectroscopic change was a direct result ofefficient FRET from the PFBT dots to the Alexa dye in close proximityand indicated the effective azide-alkyne click reaction. In addition, wealso clicked the azido-Pdots onto alkyne-functionalized silicananoparticles to convert the optically inert silica particles intohighly fluorescent probes. The Pdot-silica conjugates were clearlyvisible at the single-particle level even on a mercury lamp-illuminated,low-magnification (4×) fluorescent microscope (FIG. 17e ).

Alkyne-Alexa 594 dye was purchased from Invitrogen (Eugene, Oreg., USA)for click reactions with azido-Pdots. In a typical reaction, 50 nMazido-Pdots in MilliQ water containing 1% BSA were mixed with 5 μMalkyne-Alexa 594 dye in the presence of 1 mM CuSO₄ and 5 mM sodiumascorbate for 30 minutes before spectroscopic measurements. For clickreaction of azido-Pdots to alkyne-silica particles, silica colloids(˜200 nm in diameter) were prepared according to the standard Stobermethod. Alkyne functionalization to silica particles was performed asfollows: 80 mg of silica particles was washed with anhydrous ethanol,dried, and resuspended in 4 mL anhydrous dimethylformamide (DMF). 80 μLof O-(Propargyloxy)-N-(Trimethoxysilylpropyl)Urethane was added to thesilica in DMF suspension, and the mixture was magnetically stirred on a90° C. hotplate for 24 hours. The alkyne-functionalized silicananoparticles were washed thoroughly with ethanol, and resuspended inMilliQ water. For a typical click reaction, 0.5 mL of 50 nM azido-Pdotsin MilliQ water containing 1% BSA was mixed with 0.1 mL of alkyne-silicaparticles (20 mg/mL) in the presence of 1 mM CuSO₄ and 5 mM sodiumascorbate for 2 hours. The Pdot-decorated silica particles were thenwashed thoroughly with MilliQ water. A drop of the dilute solution ofPdot-decorated silica particles was placed on a coverslip and viewed onan upright microscope with an AZ-Plan Apo 4× objective (Nikon AZ100,Melville, N.Y., USA) and with a mercury lamp as the excitation source.

Example 21: Metabolic Labeling with Clickable Pdots

To demonstrate cellular labeling with Pdots and click chemistry, newlysynthesized proteins modified by bioorthogonal non-canonical amino-acidtagging (BONCAT) were visualized. In the BONCAT technique, newlysynthesized proteins in cells are metabolically labeled with an azido-(or alkyne-) bearing artificial amino acid. The artificial amino acidendows the proteins with unique chemical functionality that subsequentlycan be tagged with exogenous probes for detection or isolation in ahighly selective manner (D. C. Dieterich, A. J. Link, J. Graumann, D. A.Tirrell, E. M. Schuman, Proc. Natl. Acad. Sci. USA 2006, 103, 9482).Azidohomoalanine (AHA) and homopropargylglycine (HPG) are two artificialamino acids commonly used in this method. They are effective surrogatesfor methionine, an essential amino acid; in the absence of methionine,the cellular synthesis machinery straightforwardly incorporates theminto proteins. This approach is operationally similar to the traditionalmetabolic labeling with radioactive amino acid ³⁵S-methionine. Afterincorporation, AHA and HPG are susceptible to tagging with exogenousprobes, which in the present example are the highly fluorescent Pdotsfor in situ imaging.

First, AHA-labeled proteins were targeted using Pdot-alkyne probes.MCF-7 human breast cancer cells were grown to confluence before passageinto serum-free medium lacking methionine. After incubation to depleteany residual methionine, cell cultures were supplemented with AHA forfour hours. Then the cells were washed and fixed before carrying out theclick reaction with alkyne-Pdots in the presence of CuSO₄, a reducingagent (sodium ascorbate), and a triazole ligand. The Pdot-tagged cellswere viewed immediately on a confocal fluorescence microscope. Identicalsettings were used to acquire images from the Pdot-labeled cells and thenegative controls.

Briefly, for metabolic labeling of newly synthesized proteins,homopropargylglycine (HPG) and BlockAid™ blocking buffer were purchasedfrom Invitrogen (Eugene, Oreg., USA). Azidohomoalanine (AHA) waspurchased from Medchem Source LLP (Federal Way, Wash., USA). MCF-7 cellswere grown to confluence before passage into serum-free medium lackingmethionine. After one hour incubation to deplete any residualmethionine, cultures were supplemented with 0.1 mM AHA or HPG for fourhours. The cells were washed by 1×PBS, fixed with 4%paraformaldehyde/PBS, and blocked in the BlockAid™ blocking buffer. TheAHA- or HPG-labeled cells were incubated for one hour with a mixture of1 mM CuSO₄, 5 mM sodium ascorbate, 0.5 mM trisq1-benzyl-1H-1,2,3-triazol-4-yL)methypamine (TBTA, triazole ligand), and50 nM alkyne-Pdots (for AHA-labeled cells) or azido-Pdots (forHPG-labeled cells). The Pdot-tagged cells were then counterstained withHoechst 34580 and imaged immediately on a fluorescence confocalmicroscope (Zeiss LSM 510).

FIG. 18 shows confocal fluorescence and bright-field images of thePdot-labeled cells and the control samples. Very bright fluorescence wasobserved for the AHA-labeled cells tagged with Pdot-alkyne via clickreaction (FIG. 18a-18d ). When the cells were incubated under identicalconditions but in absence of the reducing agent (sodium ascorbate) thatforms copper (I) from CuSO₄, cell labeling by Pdots was not observed,indicating that Pdot-alkyne was selective for the copper (I)-catalyzedreaction (FIG. 18e-18h ). In a different control, copper (I)-catalyzedPdot-alkyne tagging was performed under identical conditions as those inFIG. 18a-18d but in cells not exposed to AHA. In this control, celllabeling also was not observed (FIG. 19), indicating Pdot-alkyne taggingwas highly specific for the cellular targets of interest. In addition,Pdot-azide was used to detect newly synthesized proteins in MCF-7 cellsincubated with HPG. In this case, the Pdot-azide also specifically andeffectively labeled the targets (FIG. 20). In comparison with thePdot-alkyne labeling (AHA-treated cells), an obvious difference in thefluorescence brightness of the Pdot-azide labeling (HPG-treated cells)was not observed. This is consistent with the literature results thatHPG and AHA show very similar activities in the synthesis of nascentproteins in mammalian cells.

Example 22: Glycoprotein Labeling with Clickable Pdots

Pdot-alkyne was used to selectively target glycoproteins, a subset ofproteins extensively involved in various biological functions. Thebioorthogonal chemical reaction strategy has been previously developedfor probing glycans on cultured cells and in various living organisms(see, for example, J. A. Prescher, D. H. Dube, C. R. Bertozzi, Nature2004, 430, 873; D. H. Dube, J. A. Prescher, C. N. Quang, C. R. Bertozzi,Proc. Natl. Acad. Sci. USA 2006, 103, 4819; S. T. Laughlin, C. R.Bertozzi, Proc. Natl. Acad. Sci. USA 2009, 106, 12; and M. A.Breidenbach, J. E. G. Gallagher, D. S. King, B. P. Smart, P. Wu, C. R.Bertozzi, Proc. Natl. Acad. Sci. USA 2010, 107, 3988). The methodinvolves metabolic labeling of glycans with a monosaccharide precursorthat is functionalized with an azido group, after which the azido sugarsare covalently tagged with imaging probes. MCF-7 cells were incubatedwith N-azidoacetylgalactosamine (GalNAz) for three days in order toenrich O-linked glycoproteins with the azido groups. The GaINAz-treatedcells were tagged with Pdot-alkyne via click reaction and subsequentlyviewed on a confocal microscope. Bright cell-surface labeling wasobserved for the cells positively tagged with Pdot-alkyne (FIG. 21a-21d). In the negative control, where cells were incubated with Pdot-alkynein the absence of the reducing agent, cell labeling was not observed(FIG. 21e-21h ). As an additional control, Pdot tagging was performedunder identical conditions but in cells lacking azides; in this case,cell labeling was not observed, again indicating Pdot labeling washighly specific for the cellular targets of interest.

Briefly, for metabolic labeling of glycoproteins,N-azidoacetylgalactosamine (GalNAz) was purchased from Invitrogen(Eugene, Oreg., USA). MCF-7 cells were cultured using the general EMEMmedium containing 50 μM N-azidoacetylgalactosamine (GalNAz) for threedays in order to enrich the azido groups in O-linked glycoproteins. TheGalNAz-labeled cells were washed by 1×PBS, fixed with 4%paraformaldehyde/PBS, and blocked in the BlockAid™ blocking buffer. Thenthe GalNaz-labeled cells were incubated for one hour with a mixture of 1mM CuSO₄, 5 mM sodium ascorbate, 0.5 mM tris((1-benzyl-1H-1,2,3triazol-4-yl)methyl)amine (TBTA, triazole ligand), and 50 nMalkyne-Pdots. The Pdot-tagged cells were then counterstained withHoechst 34580 and imaged immediately on a fluorescence confocalmicroscope (Zeiss LSM 510).

Example 23: Preparation and Functionalization of Red-Emitting PBdots

The red-emitting semiconducting polymers PF-0.1TBT and PF-0.1DHTBT weresynthesized and characterized as in previous reports (Hou, Q. et al. J.Mater. Chem. 12, 2887-2892 (2002); and Hou, Q. et al. Macromol. 37,6299-6305 (2004)). The light-harvesting semiconducting polymerpoly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}](PFPV, MW 220,000, polydispersity 3.1), andpoly[(9,9-dioctylfluorenyl-2,7-diye-co-(1,4-benzo-{2,1′,3}-thiadiazole)](PFBT, MW 157,000, polydispersity 3.0) was purchased from ADS Dyes, Inc.(Quebec, Canada). The amphiphilic functional polymerpoly(styrene-co-maleic anhydride) (PSMA, cumene terminated, averageMn˜1,700, styrene content 68%) was purchased from Sigma-Aldrich (St.Louis, Mo., USA)). All other reagents for PBdot preparation werepurchased from Sigma-Aldrich (St. Louis, Mo., USA).

Functionalized PBdots in aqueous solution were prepared by using amodified nano-precipitation method (Wu, C., Peng, H., Jiang, Y. &McNeill, J. J. Phys. Chem. B 110, 14148-14154 (2006); and Wu, C., Bull,B., Szymanski, C., Christensen, K. & McNeill, J. ACS Nano 2, 2415-2423(2008)). All experiments were performed at room temperature unlessindicated otherwise. In a typical preparation, the light-harvestingpolymer PFBT, red-emitting polymer PF-0.1TBT, and amphiphilic functionalPSMA were first dissolved in tetrahydrofuran (THF) to make a 1 mg/mLstock solution, respectively. The three polymer solutions were dilutedand mixed in THF to produce a solution mixture with a PFBT concentrationof 50 μg/mL, a PF-0.1TBT concentration of 30 μg/mL, and a PSMAconcentration of 20 μg/mL. The mixture was sonicated to form ahomogeneous solution. A 5 mL quantity of the solution mixture wasquickly added to 10 mL of MilliQ water in a vigorous bath sonicator. TheTHF was removed by nitrogen stripping. The solution was concentrated bycontinuous nitrogen stripping to 5 mL on a 90° C. hotplate followed byfiltration through a 0.2 micron filter. During nanoparticle formation,the maleic anhydride units of PSMA molecules were hydrolyzed in theaqueous environment, generating carboxyl groups on PBdots. The PBdotdispersions were clear and stable for months without signs ofaggregation.

Example 24: Surface Bioconjugation to Carboxyl Blended Polymer Dots(PBdots)

For biomolecular conjugation, tumor-specific peptide ligand chlorotoxin(CTX) was purchased from Alomone Labs, Ltd. (Jerusalem, Israel).Streptavidin was purchased from Invitrogen (Eugene, Oreg., USA).1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and amineterminated polyethylene glycol (Methyl-PEG₈-NH₂) was purchased fromThermo Fisher Scientific (Rockford, Ill., U.S.A).

Bioconjugation was performed by utilizing the EDC-catalyzed reactionbetween carboxyl PBdots and the respective amine-containing biomolecules(Methyl-PEG_(8,)—NH₂, chlorotoxin, and streptavidin). In a typicalconjugation reaction, 60 μL of polyethylene glycol (5% w/v PEG, MW 3350)and 60 μL of concentrated HEPES buffer (1 M) were added to 3 mL ofcarboxyl PBdot solution (50 μg/mL in MilliQ water), resulting in a PBdotsolution in 20 mM HEPES buffer with a pH of 7.3. Then, 30 μL ofamine-containing molecules (1 mg/mL) was added to the solution and mixedwell on a vortex. Last, 60 μL of freshly-prepared EDC solution (5 mg/mLin MilliQ water) was added to the solution, and the above mixture wasmagnetically stirred for 4 hours at room temperature. Finally, theresulting PBdot-CTX and PBdot-PEG conjugates were separated from freemolecules by Bio-Rad EconoPac® 10DG columns (Hercules, Calif., USA).PBdot-streptavidin bioconjugates were separated by gel filtration usingSephacryl HR-300 gel media.

Example 25: Characterization of Blended Polymer Dots (PBdots)

Single-particle imaging was performed to carry out a side-by-sidebrightness comparison of the PB dot with the Qdot that emits at 655 nm,the brightest one of different colored Qdot probes. With a 488 nm laserexcitation power so that Qdot 655 can be reasonably detected (FIG. 26b), the majority of PBdots actually saturated the detector underidentical acquisition and laser conditions. Such a prominent contrast isattributed to the high molar extinction coefficient of PBdots (˜3.0×10⁷cm⁻¹M⁻¹ at 488 nm for nanoparticles of ˜15 nm diameter). To avoiddetector saturation, a neutral density filter (optical density of 1,which blocks 90% of the emitted fluorescence) was placed together withthe emission filter to obtain single-particle fluorescence images of PBdots (FIG. 26c ). Thousands of particles are collected and theirfluorescence intensities were back-calculated according to theattenuation factor. Fluorescence intensity distribution indicated thatPB dot were ˜15 times brighter than Qdot 655 nanocrystals (FIG. 26d ),consistent with the brightness comparison based on the bulk spectraanalysis. Fluorescence lifetime of PBdots was determined to be 3.5 ns bya TCSPC setup. It is worth noting that single PBdots contain multipleemitters, which results in photon emission rates that are higher thanthose predicted from fluorescence lifetime alone.

Example 26: Preparation and Characterization of Conjugated PBdots

Functionalized PBdots in aqueous solution were prepared by using amodified nano-precipitation method (Veiseh, O. et al., Cancer Res. 69,6200-6207 (2009); and Choi, H. S. et al., Nature Biotechnol. 25,1165-1170 (2007)). In a typical preparation, light-harvesting polymerPFBT, red-emitting polymer PF-0.1TBT, and functional polymer PSMA weredissolved in tetrahydrofuran (THF) to produce a solution mixture withPFBT concentration of 50 μg/mL, PF-0.1TBT concentration of 30 μg/mL, andPSMA concentration of 20 μg/mL. The mixture was sonicated to form ahomogeneous solution. A 5 mL quantity of the solution mixture wasquickly added to 10 mL of MilliQ water in a bath sonicator. The THF wasremoved by nitrogen stripping. The solution was concentrated bycontinuous nitrogen stripping to 5 mL on a 90° C. hotplate followed byfiltration through a 0.2 micron filter.

Chlorotoxin (CTX), a 36-amino acid peptide, was selected as atumor-targeting ligand because of its strong affinity for tumors ofneuroectodermal origin. It has been shown that CTX specifically binds toglioma, medulloblastoma, prostate cancer, sarcoma, and intestinalcancer. First, the PBdots were functionalized by an amphiphilic polymer,poly(styrene-co-maleic anhydride) (PSMA). The hydrophobic polystyreneunits of PSMA molecules were anchored inside the PBdot particles, whilethe maleic anhydride units localized to the PBdot surface and hydrolyzedin the aqueous environment to generate carboxyl groups. The carboxylgroups enabled surface conjugations by the standard carbodiimidechemistry (Hermanson, G. T. Bioconjugate Techniques (Academic Press, SanDiego, 2008)).

Besides CTX, polyethylene glycol (PEG) can be conjugated to reduceprotein adsorption, limit immune recognition, and thereby increase thenanoparticle serum half-life in vivo. Streptavidin was also used inbioconjugation as a separate control to verify the conjugation strategyby specific cellular labeling. Transmission electron microscopy (TEM)showed that both the bare and the functionalized PBdots had comparableparticle sizes (˜15 nm in diameter) (FIG. 26d ), consistent with thedynamic light scattering results (FIG. 29). After conjugation todifferent molecules (PEG, CTX, and streptavidin), gel electrophoresisshowed shifted migration bands of the PBdot-conjugates in a 0.7% agarosegel due to the changes in surface charge and particle size. Theseresults clearly show successful carboxyl functionalization and surfacebioconjugations.

Surface bioconjugation was performed by utilizing the EDC-catalyzedreaction between carboxyl Pdots and the respective amine-containingbiomolecules (chlorotoxin, Methyl-PEG₈-NH₂, or strepavidin). In atypical conjugation reaction, 60 μL of polyethylene glycol (5% w/v PEG,MW 3350) and 60 μL of concentrated HEPES buffer (1 M) were added to 3 mLof carboxyl PBdot solution (50 μg/mL in MilliQ water), resulting in aPdot solution in 20 mM HEPES buffer with a pH of 7.3. Then, 30 μL ofamine-containing biomolecules (1 mg/mL) was added to the solution andmixed well on a vortex. Last, 60 μL of freshly-prepared EDC solution (5mg/mL in MilliQ water) was added to the solution, and the above mixturewas magnetically stirred for 4 hours at room temperature. Finally, theresulting PBdot-CTX and PBdot-PEG conjugates were separated from freemolecules by Bio-Rad Econo-Pac® 10DG columns (Hercules, Calif., USA).PBdot-streptavidin bioconjugates were separated by gel filtration usingSephacryl HR-300 gel media.

The long-term fate and in vivo stability are of both fundamental andclinical significance for designing in vivo probes. The integrity ofnanoprobes is primarily dependent on their chemical reactivity towardsionic species and reactive oxygen species (ROS) in biologicalenvironment. For example, Qdots undergo severe chemical degradation dueto copper ions and ROS at physiological concentrations, which cause theloss of luminescence and release of toxic Cd ions. The sensitivity ofPBdots to pH, biologically relevant ions, and ROS was examined. PBdotsshowed constant fluorescence in physiological pH range from 4 to 9 (FIG.30). The fluorescence of PBdots was also not affected by anybiologically relevant ions under test (FIG. 31), including iron, zinc,and copper, three of the most abundant ions in biological organisms.Furthermore, the two common and stable ROS in physiological environment,hypochlorous acid (HOCl) and hydrogen peroxide (H₂O₂), do not show anyeffect on the fluorescence of PBdots. In contrast, thestreptavidin-conjugated, polymer-encapsulated Qdot 655 probes aresignificantly quenched by H₂O₂ and iron ions, and completely quenched byHOCl and copper ions, with the same concentrations as used for PBdots.The stable fluorescence of PB dots can be attributed to theirhydrophobic organic polymeric nature, which tends not to have chemicalinteractions with the metal ions and ROS. This property provides asignificant advantage for using PBdots as in vivo probes.

Example 27: Detection of Cu²⁺ and Fe²⁺ Ions by Functionalized Pdots

Typically, 40 μg of PFBT and 8 μg of PSMA were dissolved into 5 mL ofTHF. This mixture was then quickly injected into 10 mL of water undervigorous sonication. The THF was then removed by purging with nitrogenon a 96° C. hotplate for one hour. The resulting Pdot solution wasfiltered through a 0.2 μm cellulose acetate membrane filter to removeany aggregates formed during preparation.

A highly fluorescent semiconducting polymer,poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,1′-3}-thiadiazole)](PFBT) was employed and the PSMA polymer (20%) was added into the PFBTmatrix to form the PS-COOH co PFBT Pdots. The carboxyl groups on thePdot surface were produced by the hydrolysis of the maleic anhydrideunits while the polystyrene portions tended to anchor inside thehydrophobic core of the PFBT polymer. These Pdots dispersed well inaqueous solutions and the carboxyl moieties served as selectivecoordination groups for metal ions without further modification. Thefluorescence of the carboxyl-functionalized PFBT Pdots were found to bequenched selectively by Cu²⁺ and Fe²⁺ ions (FIG. 32).

Several different types of polymers, including PFBT andpoly[(4-(5-(7-methyl-9,9-dioctyl-9H-fluoren-2-yl)thiophen-2-yl)-7-(5-methylthiophen-2yl)benzo[c][1,2,5]thiadiazole)](PFTBT) Pdots (without being blended with PSMA), exhibited excellentstability (no aggregation and quenching) toward various ions in buffersolution (vide infra), which demonstrates that unfunctionalized Pdotscan serve as a good control in these methods.

The aggregation and self-quenching behavior of Pdots was attributed tothe chelating interactions between the PS-COOH groups on the surface ofPdots with Cu²⁺ and Fe²⁺ in solution. We note we did not add PEG intothe solution in the experiments described in this example, because PEGcan prevent the aggregation and self-quenching behavior of Pdots. Thisphenomenon is clearly revealed by transmission electron microscopy (TEM)measurements taken before and after the addition of Cu²⁺ (FIGS. 33A andB). Dynamic light scattering (DLS) measurements also showed that thediameter of the as-prepared Pdots was ˜21 nm on average before theaddition of Cu²⁺, but increased to ˜500 nm after the addition of Cu²⁺(FIG. 33C).

The effect of various other physiologically important cations on thefluorescence of PS-COOH co PFBT Pdots was also investigated. It wasfound that their emission intensity was unaffected or minimally affectedby other cations, as compared with the emission intensity of PS-COOH coPFBT Pdots in pure water (I_(blank)) (FIG. 34).

In this study, non-functionalized PFBT co PFTBT Pdots were used as aninternal standard. While PS-COOH co PFBT Pdots emit at 540 nm whenexcited at wavelengths below 490 nm, PFBT co PFTBT Pdots emit at 623 nmwhen excited at the same wavelengths.

This shift in emission wavelength is caused by the efficient energytransfer from PFBT to PFTBT and subsequent emission from PFTBT. The useof PFBT co PFTBT Pdots as an internal standard allowed the applicationof ratiometric ion determination based on two fluorescence emissionintensities (540 nm/623 nm), thereby eliminating any interference fromthe environment or drift in the instrument.

FIG. 35A shows the emission spectra of solutions containing PS-COOH coPFBT Pdots and PFBT co PFTBT Pdots as a function of Cu²⁺ concentration.It is evident that while the emission intensity of the PFTBT containingPdots (623 nm) remained constant, the PS-COOH containing Pdots decreasedtheir intensity (540 nm) with increasing Cu²⁺ concentrations up to 30μM. No further decrease in fluorescence intensity was observed forconcentrations above 30 μM, indicating that all of the carboxyl groupshave already been occupied by copper ions. For this set of experiments,the relative standard deviation of blank signal from 10 replicates was1.1%. A quenching signal of ˜6% could be observed at 100 nM of copperions. FIG. 35B shows a linear correlation between the ratio ofΔI_(540 nm)/I_(623 nm) and Cu²⁺ concentration, which ranged from 1 μM to30 μM (R²=0.992).

Because the aggregation-induced fluorescence quenching was caused by theformation of 2:1 sandwich complexes between carboxyl moieties on thePdot surface and Cu²⁺, one might expect that a strong copper ionchelator, ethylenediaminetetraacetic acid (EDTA), might be able toprevail over carboxyl groups and thus redisperse the aggregation. Totest this hypothesis, the same molar amount of EDTA as Cu²⁺ ions wasadded into the aggregated Pdot solution and found that the fluorescenceintensity of PS-COOH co PFBT Pdots was completely restored (FIG. 35C).

Moreover, the addition of excessive amounts of EDTA into the solutiondid not lead to any further increases in emission intensity, whichsuggests that no additional copper ion could be chelated by EDTA. TheDLS experiments also verified that the aggregated Pdots were redispersedafter the addition of EDTA (FIG. 33C).

Notably, this process could be repeated many times with minimal relativesignal loss, which indicates that this protocol could be reused for manycycles rather than just as a one-time-use assay. This reversibility wasnot observed on quantum dot-based (e.g. CdSe) Cu²⁺ sensors due to thenon-reversible cation-exchange processes between Cu²⁺ and Cd²⁺ (Y.-H.Chan Y. H. et al., Anal. Chem., 2010, 82, 3671-3678; and Sadtler B. etal., J. Am. Chem. Soc., 2009, 131, 5285-5293). It is worth noting thatthe response time for both the aggregation and redispersion of Pdots wasvery short (less than 1 min) and the fluorescence intensity remainedunchanged for several days after reaction.

The aggregation and self-quenching phenomenon could be observed also forFe²⁺ sensing with a dynamic range from 10 to 25 μM as shown in FIG. 36(R²=0.996). The fluorescence intensity of PS-COOH co PFBT Pdots wasquenched significantly (by as much as 70%) by Fe²⁺, but this quenchingcould not be reversed by adding EDTA, which is likely due to the muchlower binding constant of EDTA for Fe²⁺ than for Cu²⁺.

We also adjusted the pH value of the Pdot-Fe²⁺ mixture in an effort toprotonate the carboxyl groups, and then added excessive amounts of EDTAinto the solution. However, no substantial emission intensity (less than10%) could be recovered even at pH=1. Besides, these Pdots are prone toaggregation at pH below 2. Therefore, the selective recovery offluorescence, by addition of EDTA, from Cu²⁺-induced self-quenchingallowed us to differentiate between copper and iron sensing and todetermine their concentrations individually.

Example 28: Simultaneous Detection of Cu²⁺ and Fe²⁺ Ions byFunctionalized Pdots

To demonstrate the application of the detection method outlined inExample 26, cell-culture media was selected because it simulates complexphysiological fluids and at the same time also serves as a wellcontrolled solution. Here, the test samples were prepared by spiking 10μM and 15 μM of copper and iron ions, respectively, into Dulbecco'sModified Eagle Medium (DMEM D-5921) solutions containing the Pdotsensors. Then EDTA was added into the solution so that the concentrationof Cu²⁺ could be calculated from the restored emission intensity ofPdots. We then estimated the Fe²⁺ concentration by comparing to theI_(blank). This measurement showed that the concentrations of copper andiron were 10.17±1.34 μM and 16.16±1.82 μM, respectively, which exhibitedgood accordance with the spiking values. This experiment demonstratesthe feasibility of using this Pdot-based sensing system for copper andiron detection in complex samples.

Example 29: Functionalized Chromophoric Polymer Dots with Near InfraredFluorescence

This example provides a demonstration that near infrared (NIR) dyes canbe doped into the functionalized chromophoric polymer dot (CPdot orPdot) to tune the fluorescence properties of the chromophoric polymerdot bioconjugate. We use below CPdots or Pdots interchangeably todescribe chromophoric polymer dots.

Preparation and characterization of NIR dye-doped CPdots. In a typicalprocedure, a THF solution containing 50 μg/mL of PFBT, 50 μg/mL ofPS-PEG-COOH, and 0.2 μg/mL of a NIR dye, silicon 2,3-naphthalocyaninebis(trihexylsilyloxide) (NIR775) was prepared. A 5 mL aliquot of themixture was then quickly dispersed into 10 mL of water under vigoroussonication. The extra THF was evaporated at an elevated temperature(lower than 100° C.) with the protection of nitrogen gas. The THF-freeCPdot solution was filtrated through a 0.2 μm cellulose membrane filterand adjusted to the appropriate concentration. The size and themorphology of the CPdots were investigated using a transmission electronmicroscope (PEI Tecnai F20, 200 kV). The size of the CPdots was alsomeasured in aqueous solution using a dynamic light scattering instrument(Malvern Zetasizer NanoZS). UV-Vis absorption spectra of CPdots and NIRdyes were recorded with a DU 720 scanning spectrophotometer (BeckmanCoulter, Inc., CA USA) in water. The carboxyl surface of CPdots wasverified by testing the Zeta potential using Malvern Zetasizer NanoZS.CPdots with and without carboxyl surface were both tested in a gelelectrophoresis experiment. The gel was prepared using 0.7% of normalmelting agarose, 0.2% of PEG (MW 3350) and 20 mM HEPES buffer. The CPdotsamples were loaded to electrophoresis channels with the help of 30%glycerol, and run in 20 mM HEPES buffer (pH 7.4) under anelectrophoresis force of 10V/cm for 15 min using a Mupid®-exU submarineelectrophoresis system. The gel was then developed using a Kodak imagestation 440CF system. Fluorescence spectra of CPdots and NIR dyes weremeasured using a Fluorolog-3 fluorospectrometer (HORIBA Jobin Yvon, NJUSA). Fluorescence lifetime data of NIR dye-doped CPdots and PFBT dotswere obtained using a time-correlated single-photon counting instrument(TCSPC). Fluorescence quantum yields of PFBT dots and NIR dye-dopedCPdots were collected by an integrating sphere (Model C9920-02,Hamamatsu Photonics) with a 457 nm excitation from a 150 W CW Xenonlamp.

Functionalized CPdots with NIR emission. We formulated the NIR dye-dopedCPdots using three components of different functions: greensemiconducting polymer (PFBT), NIR dye (NIR775), and amphiphilic polymer(PSPEG-COOH) (FIG. 37). As the essential part of CPdots, thesemiconducting polymer formed the hydrophobic matrix of Pdot and servedas a host for the NIR dyes. NIR775 dyes are highly fluorescent inhydrophobic solvents while they are significantly less so inphysiological environments due to self-aggregation. The aggregation wasavoided or significantly reduced when these dyes were doped inside Pdotmatrices. Inside the CPdots, the NIR dyes serve as an acceptor receivingenergy from the semiconducting polymer matrix, and generate strong NIRfluorescence. For the purpose of fluorescence imaging, the surface ofCPdots was modified with carboxyl groups using amphiphilic polymer,PS-PEG-COOH. Here, the hydrophobic part of the polymer entangled withPdot matrix and the hydrophilic part stretched out into thephysiological environment. The NIR dye-doped CPdots were synthesizedusing the nano-precipitation method because of its simplicity and highefficiency for Pdot fabrication and hydrophobic dye doping (FIG. 37).All the components of CPdots were first dissolved and mixed in anhydrousTHF, and then quickly precipitated in water under sonication. The suddenchange of solvent environment and the strong sonicating force produced ananometer-sized semiconducting polymer particle and simultaneouslytrapped the hydrophobic NIR dye (NIR775) in the CPdot matrix. Thecarboxylate surface was also generated as the amphiphilic polymerassembled at the interface of CPdots and water.

Both the TEM image and the DLS results show that the NIR dye-dopedCPdots were monodispersed particles with an average diameter of 18 nm(FIGS. 38A and 38B). In these particles, NIR dyes were successfullyencapsulated as proven by the following experiments. First, the Pdotsolution was filtrated using a 100K molecular weight cutoff centrifugalmembrane, which only allows free NIR dyes to pass through but not thedoped ones. The filtrate did not contain NIR dye as monitored by UV-Visspectrometer, while the absorbance of NIR dye was observed in thesolution of the NIR dye-doped CPdots after the filtration. This resultshows NIR dyes were completely doped in the CPdots. The surface ofCPdots was functionalized with carboxylate groups using the amphiphilicpolymer PSPEG-COOH. This surface functionalization significantlydecreased the zeta potential of the CPdots from ˜35.4 mV (bare PFBTDots) to −46.0 mV (CPdots of PS-PEG-COOH coating). Gel electrophoresisalso showed the carboxylate CPdots moved much faster than the bare PFBTDots of similar size towards the positive electrode (FIG. 38C). The NIRdoping did not affect the surface potential of the carboxylate CPdots,which suggested that NIR dyes were only located inside the CPdots butnot on the surface.

Energy transfer mediated fluorescence in NIR Dye-Doped CPdots. Excitedat 457 nm, the NIR dye-doped CPdots possess two fluorescence emissions:one visible emission from the polymer matrix and one NIR emission fromthe doped NIR dyes (FIG. 39). The two fluorescence emissions weremodulated by manipulating the NIR dye concentration inside the Pdot. NIRdye can efficiently quench the CPdot fluorescence even at a lowconcentration; the reduction of the polymer fluorescence was achieved byincreasing the NIR dye concentration in the range from 0.2% to 2% (FIG.40A). The NIR emission of the CPdot was also modulated by controllingthe concentration of dye doping. Doping more NIR dyes into CPdot matrixlead to a drop of the NIR fluorescence because NIR dyes may self quenchin CPdots at high concentrations (FIG. 40E). Therefore, there is anoptimal doping concentration for maximizing the fluorescence in the NIRregion.

There is efficient intra-particle energy transfer from PFBT polymer toNIR dyes. Even a small amount of NIR dye (0.2% w/w) was adequate toquench the polymer fluorescence by 75 percent. More than 95 percent ofthe polymer fluorescence was quenched when 2% of NIR dye was doped. Thequenching results were well described by Stern-Volmer relationship (FIG.40D). The quenching of Pdot matrix was also evident by the change of thefluorescence lifetime of the CPdots before and after NIR dye doping. Thelifetime of the PFBT dots, which was originally 2.4 ns, decreased to 1.2ns after doping 0.2% of NIR dye into Pdot matrix. The fluorescencequantum yield of the 540 nm emission also dropped from 0.368 to 0.08according to the NIR dye doping. The NIR dye-doped CPdots canefficiently convert the received energy to NIR emission by transferringenergy from the matrix to the NIR dyes. For example, the 0.2% NIRdye-doped CPdots exhibited a strong NIR emission, which is comparable tothe 540 nm peak of the CPdots without dye doping. The quantum yield ofthis NIR emission was around 0.11, which indicates an efficient energyconversion. The fluorescence intensity of the NIR dye was greatlyenhanced by the doping strategy. Excited at 457 nm in the aqueoussolution, the doped NIR dye exhibited 40 times stronger fluorescenceover the equivalent amount of free NIR dye excited at 763 nm in THE(FIG. 41A).

The light harvesting efficiency is one key parameter that determines thefluorescence brightness of nanoparticles. Doping NIR dyes into CPdotextensively improved the light harvesting capability of the doped dyes.The brightness of the NIR dye-doped CPdots is compared with NIR quantumdots (Qdot800 from Invitrogen Inc.). At the same particle concentration,NIR dye-doped CPdots are about 4 times more intense and have muchnarrower fluorescence emission than Qdot800 (FIGS. 41B and 41C).

Dye-leakage. To exam the dye leakage, the acceptor-to-donor fluorescenceratio of the NIR dye-doped CPdots was monitored in 20 mM HEPES bufferfor 72 hours. The results show that the acceptor-to-donor ratio onlyslightly decreased to 85% after 72 hours, and the NIR fluorescence didnot change (FIG. 42A). This result indicates that the hydrophobic NIRdyes are unlikely to leak out to aqueous solutions and the NIR dye-dopedCPdots can sustain their fluorescence properties for at least 72 hoursand likely much longer. Because the NIR dye-doped CPdots are desirablefor the in vivo applications, the leaking test was also carried out inhuman plasma at 37° C. The result shows similar data to the former testat room temperature, which suggests that the change of the solutionconditions does not compromise the performance of the NIR dye-dopedCPdots in 72 hours (FIG. 42B). To completely overcome dye leakage, wecan covalently link the dye molecules to the chromophoric polymer matrixto form CPdots.

Example 30: Functionalized Chromophoric Polymer Dots for RatiometricTemperature Sensors

This example provides a demonstration that functionalized chromophoricdots can be used to form ratiometric nanoparticle temperature sensors.We use below CPdots or Pdots interchangeably to describe chromophoricpolymer dots.

Preparation of CPdot temperature sensor. First, we use functionalpolymer such as amine-terminated polystyrene polymer to react with atemperature sensing dye Rhodamine B (RhB). Because Rhodamine B is awater-soluble dye, the reaction with a functional hydrophobic polymercan make the dye hydrophobic, therefore it can be entrapped inside thehydrophobic CPdots. In a 10 mL round-bottom flask, 200 μL of 10 mg/mLRhodamine B-isothiocyanate in DMF (anhydrous) and 1 mg NaHCO₃ were addedinto 2 mL 1 mg/mL (in DMF) amine-terminated polystyrene (PS-NH₂, MW1000, polydispersity 1.1) to perform NH₂-isothiocyanate reaction asshown in FIG. 43A. The mixture was gently stirred overnight under theprotection of N₂. DMF was removed by rotary evaporation at 75° C. Theresulting red solid was then dissolved in 1 mL THF (anhydrous). NaHCO₃was filtered off with 200 nm membrane filter since it did not dissolvein THF. The obtained PS-RhB in THF was then doped into chromophoricpolymer to make CPdot.

The copolymers poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV, MW 220 000,polydispersity 3.1), and poly[(9,9dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}-thiadiazole)] (PFBT,MW 150 000, polydispersity 3.0), were used for preparation of CPdottemperature sensors. 10 mg of the above polymer was dissolved in 10 mLTHF by stirring overnight under inert atmosphere. Rhodamine B dopedCPdots were prepared by first mixing 200 μL of 1 mg/mL (in THF) PFPV orPFBT and 10 μL, of 2 mg/mL (in THF) PS-RhB in 5 mL THF and then byinjecting the mixed polymer in THF solutions into 10 mL of MilliQ waterunder sonication. THF was then removed by partial vacuum evaporation,and a small fraction of aggregates was removed by filtration through a0.2 μm membrane filter. Free Rhodamine B is removed by Bio-RadEcono-Pac® 10DG columns (Hercules, Calif., USA). FIG. 43B shows theillustration of CPdot preparation and the energy transfer between thechromophoric polymer and RhB within the nanoparticles.

Characterizations of CPdot temperature sensors. The particle size ofPdots in bulk solution was characterized by dynamic light scattering(DLS, Malvern Zetasizer NanoZS). FIG. 44 shows the DLS data for thePdots. The resulting PFPV-RhB dots are about 26 nm in diameter whilePFBT-RhB dots are 160 nm in average diameter. The CPdot size is highlydependent on the molecular weight of polymer, polymer backbone structureand the concentration ratio of PS-RhB to the chromophoric polymer. InPFBT-RhB system, 20 wt % or higher PS-RhB concentration results in theaggregation of nanoparticles. We found 10% PS-RhB in PFBT Pdot showedthe best sensitivity and relatively small size. In PFPV-RhB system, verysmall Pdots were obtained. 10% PS-RhB is also confirmed to be the bestdoping amount based on the fluorescence intensity and sensitivity.

UV-Vis absorption spectra were recorded with a DU 720 spectrophotometer.Fluorescence spectra were collected with a Fluorolog-3 fluorometer. AllCPdots were excited at 450 nm because both PFPV and PFBT showed theiradsorption peaks around 450 nm. FIG. 45A shows the absorption (dashed)and emission (solid) spectra of PFBT (black) and PFBT-RhB (red) dots atroom temperature. In the absorption spectrum, an additional smallabsorption peak at 540 nm appeared, accompanied by a 10 nm red shiftfrom 450 nm to 460 nm for PFBT absorption peak. The 540 nm peakcorresponds to the absorption of RhB, while the red-shift of PFBT is dueto the size increase from ˜20 nm of pure PFBT dot to ˜160 nm PFBT-RhBdots. PFBT-RhB dots shows two emission peaks, a relatively weak emissionpeak at 540 nm and a strong emission peak at 573 nm. The latter peakcorresponds to the emission of RhB. Because of the good overlap betweenthe emission of PFBT (peak at 540 nm) and the adsorption of RhB (peak at540 nm) as well as the close proximity between PFBT polymer chain andRhodamine B molecule, efficient energy transfer resulted in strongfluorescence of RhB, whereas pure RhB dye was poorly excited by 450 nm.

FIG. 45B shows the absorption and emission spectra of PFPV and PFPV-RhBdots at room temperature. In the absorption spectrum, PI-PV-RhB dotsshow a small absorption peak at 540 nm for Rhodamine B, while there wasno noticeable shift for PFPV peak at 450 nm. The emission spectrum showstwo peaks at 510 nm and 540 nm for PFPV dots, while PFPV-RhB dot showsan additional RhB peak at 573 nm. Similar to PFBT-RhB, there is strongFRET between PFPV and RhB due to the good spectral overlap between theemission of PFPV (peak at 540 nm) and the adsorption of RhB (peak at 540nm) and the compact packing of polymer chain and RhB inside thenanoparticle.

Fluorescent CPdot temperature sensing. The temperature-dependentfluorescence of Pdots was measured in a Fluorolog-3 fluorometer couplingwith a heating/cooling system. The absolute temperature of Pdotsolutions was measured by digital thermometer TM902C by inserting atemperature probe into the solution. The solution is stirred gently toyield homogeneous cooling and heating during the experiment. FIG. 46shows the fluorescence spectra of PFBT-RhB (A) and PFPV-RhB (B) towardstemperature rising from 10° C. to 70° C. and 10° C. to 60° C.,respectively. The right dashed line (red) shows the emission peak of RhBat 573 nm, while the left one is 510 nm which is selected as internalreference for the ratiometric calculation. FIG. 47 shows thefluorescence intensity as a function of temperature for both Pdots. Thefluorescence intensity decreases for PFBT-RhB dot as temperatureincreases. More importantly, the intensity shows a linear relationshiprelative to temperature change. The unique characteristics of theseCPdot sensors over free Rhodamine B or other nanoparticles astemperature probe are the ratiometric measurements. FIG. 48 showsratiometric plots of I_(573 nm)/I_(510 nm) as a function of temperaturefor PFBT-RhB dot (A) and PFPV-RhB dot (B). Linear fittings welldescribed the sensing behavior, so the temperature can be determinedfrom a given fluorescence intensity ratio obtained in experiment.

Example 31: Functionalized Chromophoric Polymer Dots for Ratiometric pHSensors

This example provides a demonstration that functionalized chromophoricdots can be used to form ratiometric nanoparticle pH sensors. We usebelow CPdots or Pdots interchangeably to describe chromophoric polymerdots.

Preparation of CPdot pH sensors. We used a chromophoric polymerpoly(2,5-di(3′,7′-dimethyloctyl)phenylene-1,4-ethynylene (PPE) as thepolymer donor and a pH sensitive dye fluorescein as a acceptor to formratiometric CPdot pH sensors. Thiol-terminated polystyrene (PS-SH) oramino-terminated polystyrene (PS-NH₂) were used to covalently linked thedye to CPdots.

Preparation of Thiol Functionalized PPE dots. Typically, 40 μg of PPEand 12 μg of PS-SH were dissolved into 5 mL of THF. This mixture wasthen quickly injected into 10 mL of water under vigorous sonication. TheTHF was then removed by purging with nitrogen on a 96° C. hotplate forone hour. The resulting Pdot solution was filtered through a 0.2 μmcellulose acetate membrane filter to remove any aggregates formed duringpreparation.

Preparation of Fluorescein Conjugated PPE dots (Pdot(A), Pdot(B), andPdot(C)) (FIG. 49). For Pdot(A), 0.2 mg of fluorescein isothiocyanate(FITC) dissolved in anhydrous DMSO was added to a 4 mL of PS-SH co PPEPdot (20 μg/mL) aqueous solution in a glass vial. The mixture wasstirred for 12 h at room temperature and then was purified through aBio-Rad EconoPac® 10DG column (Hercules, Calif., USA) to separate fromthe free FITC molecules. For the preparation of Pdot(B), 5 mg FITC and20 mg PS-NH₂ were dissolved and mixed in 1 mL DMF. Then 5 mLtriethylamine was added to the solution. The mixture was left on arotary shaker overnight. 0.2 mL of PPE (1 mg/l mL) in THF and 27 μL ofPS-NH₂-FITC conjugate solution were mixed into 5 mL of THF. This mixturewas then quickly injected into 10 mL of water under vigorous sonication.The THF was then removed by purging with nitrogen on a 96° C. hotplatefor one hour. The resulting Pdot(B) solution was first filtered througha 0.2 μm cellulose acetate membrane filter to remove any aggregatesformed during preparation, and then purified through a Bio-Rad EconoPac®10DG column to separate from the free FITC molecules. For thepreparation of Pdot(C), 15 μg of fluorescein-5-maleimide in anhydrousDMSO, and 60 μL of concentrated HEPES buffer (1 M) were added into afreshly prepared 4 mL of PS-SH co PPE Pdot (20 μg/mL) aqueous solutionin a glass vial. The mixture was stirred for 12 h at room temperatureand was then purified through a Bio-Rad Econo-Pac® 10DG column toseparate from the free FITC molecules.

Selection and Optimization of chromophoric polymer-Fluorescein Pair. Tofabricate a ratiometric sensing platform based on FRET and excited undera single wavelength, the first step is to select a suitabledonor-acceptor pair. Herein the fluorescein was chosen as the FRETacceptor because its absorption profile and emission intensity is highlydependent on the pH. The changes in absorption profile of fluorescein atdifferent pH allow us to modulate the FRET efficiency between the donor(chromophoric polymer matrix) and the acceptor (fluorescein molecules),whereas the response of fluorescence intensity to pH provides a feedbackof proton activity. In order to optimize the FRET efficiency, it isimportant to select an ideal donor polymer based on its spectroscopicproperties. We found the PPE polymer dots exhibited a great energytransfer to fluorescein in several conjugation routes. This phenomenoncan be interpreted by the substantial spectral overlap between theemission spectrum of PPE polymer and the excitation spectrum offluorescein as shown in FIG. 50A.

Conjugation of fluorescein to PPE Pdots. The PPE Pdots were used as thepolymer matrix while the fluorescein dyes were immobilized onto PPEPdots by virtue of three different routes as depicted in FIG. 49. Forroutes A and C, we first prepared the thiol-functionalized PPE Pdots byblending the thiol-terminated polystyrene into the PPE polymer, formingthe PS-SH co PPE Pdots in aqueous solution by the precipitation method.Subsequently, the PS-SH co PPE Pdots were reacted with fluoresceinisothiocyanate (route A) or fluorescein-5-maleimide (route C),generating the pH-sensitive Pdot-dye complex. Because we foundamino-functionalized PPE Pdots to be unstable, we devised route B, wherefluorescein isothiocyanate was first coupled to the amino-terminatedpolystyrene (PS-NH₂) under organic phase and was then blended with thePPE polymer followed by the nanoparticle precipitation in aqueoussolution. We then investigated the ratiometric sensing capability ofeach system by manipulating the ratio of polymer to dye. For example, inroute A and C, we examined different blending percentage of PS-SH in thePPE polymer and found a high sensitivity with a low standard deviationcould be obtained at 30% of PS-SH doping level. Similar results werealso observed in the route B system, in which 60% of PS-NH₂-fluoresceinblending led to the best ratiometric pH sensitivity.

FRET between PPE and fluorescein. Once the fluorescein molecules wereattached onto the PPE Pdots, an efficient energy transfer from PPE tofluorescein could be readily observed. This effective FRET is partiallyattributed to the considerable spectral overlap between the emissionspectrum of PS-SH co PPE Pdots (red solid line) and the excitationspectrum of fluorescein (black dashed line) as shown in FIG. 50A. TakePdot(A) for example, as compared to the fluorescence spectrum of barePS-SH co PPE Pdots of the same concentration (without dye conjugation,red solid line), the increase of the emission of the dye (λ=513 nm, bluesolid line) with concomitant suppression of the PPE Pdot emission(2=420-490 nm, blue solid line) clearly demonstrated that the FREThappened from the Pdot to the dye. It should be noticed that theemission intensity of unbound fluorescein at the same concentration wasvery weak when excited by the same wavelength of 390 nm, meaning thatmost of the emission intensity of fluorescein arose from the energytransfer rather than the fluorescence itself. To further confirm theFRET phenomenon, time resolved fluorescence decay curves of PPE Pdots(440±20 nm) were measured for the control, PS-SH co PPE Pdots and thePdot-dye complex, Pdot(A). The fluorescence lifetime of Pdot control was0.30 ns, while the lifetime of Pdot(A) was shortened to be 0.21 ns,indicating the occurrence of FRET from the PPE Pdot to the fluorescein.To better understand the spectroscopic behaviors of the Pdot-dyecomplexes, we conducted additional studies of the calculation of FRETefficiency at different pH as described below. In addition to thespectral overlap, to ensure an efficient FRET behavior, the size of thePdot plays an important role in that the FRET depends on the inversesixth power of the intermolecular separation. The resulting hydrodynamicdiameters of the Pdot-dye complexes measured by dynamic light scattering(DLS) are 26 nm, 25 nm, and 26 nm on average for route A, B, and C,respectively. A typical transmission electron microscopy (TEM) image ofPdot(C) is as shown in FIG. 50, which is consistent with the DLSmeasurements.

pH-sensitivity and reversibility measurements. Fluorescence spectroscopymeasurements were performed in each system to study the ratiometricresponse to pH in HEPES buffer solutions. As shown in FIG. 51, theemission peak of fluorescein increased with increasing pH, while thefluorescence intensity of PPE Pdots remained constant in all of thethree systems. We also examined the pH response of PPE Pdots alone andthe result again suggests that the PPE Pdot is pH non-responsive frompH=5.0 to 8.0, rendering it a good reference for the ratiometric pHdetection. FIG. 52 shows that the emission intensity ratio offluorescein (λ=513 nm) to PPE (λ=440 nm) changes linearly as a functionof pH ranging from 5.0 to 8.0 for these three complexes. Among them,Pdot(A) reveals the highest detection sensitivity withI_(513 nm)/I_(440 nm) varying by 0.37 per unit change in pH. This highsensitivity might originate from the shortest separation between thePdot and the dye, adding an efficient energy transfer from PPE tofluorescein. Nevertheless, it is known that thiocarbamoyl unit betweenthiol and isothiocyanate is prone to gradual degradation in the presenceof excess free thiols, making Pdot(A) less feasible in more complicatedbiological applications. On the contrary, Pdot(B) provides a stableamine-isothiocyanate adduct between the Pdot and the dye but the longside chain of PS-NH₂ results in a relatively low pH sensitivity of 0.18variation per unit change in pH on account of the less efficient energytransfer. To overcome the above mentioned obstacles, Pdot(C) was aimedto offer a complementary system with good pH sensitivity and highbonding stability. The ratio of with I_(513 nm)/I_(440 nm) varied by0.29 per unit change in pH for Pdot(C) system.

All of these three nanosensors showed good pH reversibility. TakePdot(C) for example, the pH of solution containing Pdot(C) nanosensorswas varied between pH=5 and pH=8. The good reproducibility of thefluorescein to PPE fluorescence emission ratio is indicative of thegreat reversibility and robustness for this Pdot-based pH sensor (FIG.52). Additionally, the pH response time of the sensor is too fast to bemeasured by a conventional fluorescence spectrometer owing to the smallsize (i.e., large surface-to-volume ratio) of the Pdots. FIG. 53 showsthe spectral overlap between PPE and fluorescein as well as thecalculated Forster distance as a function of solution pH.

Intracellular pH Measurements. To demonstrate the applicability of thisFRET-based ratiometric Pdot-nanosensors for intracellular pHmeasurements, the Pdot-fluorescein nanoparticles were introduced intoliving HeLa cells through endocytic processes without any additionalagents. After particle uptake, the non-incorporated particles wereremoved by extensive washing with PBS buffer. FIG. 54 shows the confocalfluorescence microscopy images of HeLa cells after Pdot(A) ingestion(E-G), while the bare PPE Pdots in cells is served as a negative control(A-C). The blue channels (A&E) were obtained by integrating the spectralregion from 433-444 nm and green channels (B&F) were acquired byintegrating the fluorescence signals from 507-518 nm. The intracellularpH was determined by comparing the ratio between the averagefluorescence signal from 507-518 nm and the average fluorescence from433-444 nm to the pH calibration curve. The average pH value based on atleast 50 cells using the Pdot(A) system was estimated to be 4.95±0.70,which is in good agreement with the reported pH ranges for the acidicpathways of endocytosis, as in the case of early endosome (pH˜6.5) andlysosomal compartment (pH=4.5-5.0). The pH values measured by using thePdot(B) and Pdot(C) probe were found to be 4.81±0.86 and 4.92±0.64,respectively (FIG. 55), which again demonstrated the feasibility ofthese Pdot-based nanosensors for the intracellular pH measurements. Moreimportantly, from the amplified and overlay images as shown in thetop-right insets of FIG. 55A-C, it clearly reveals a perfectco-localization of both the fluorescence of PPE and fluorescein. Thisco-localization suggests a simultaneous uptake of PPE and fluorescein byHeLa cells rather than the individual fluorophore uptake as a result ofunstable bond breakage.

While this invention has been described with an emphasis on preferredembodiments, it will be apparent to those skilled in the art thatvarious modifications and variations can be made in the presentinvention without departing from the scope or spirit of the invention.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being defined by the following claims.

1-19. (canceled)
 20. A method for detecting biological molecules, themethod comprising: adhering a chromophoric polymer dot to a biologicalmolecule associated with a cell in a fluid, thereby forming abioconjugated chromophoric polymer dot, wherein the chromophoric polymerdot comprises a chromophoric polymer and an amphiphilic molecule,wherein: the amphiphilic molecule comprises a hydrophobic moiety and ahydrophilic moiety; the hydrophilic moiety comprises one or morereactive functional groups; the weight ratio of the amphiphilic moleculeto the chromophoric polymer is from about 1% to about 50%; and detectingthe bioconjugated chromophoric polymer dot using one or more of flowcytometry or fluorescence microscopy imaging.
 21. The method of claim20, wherein the detecting comprises flow cytometry.
 22. The method ofclaim 20, further comprising: illuminating the fluid; and detectingluminescence from the bioconjugated chromophoric polymer dot.
 23. Themethod of claim 22, wherein the luminescence is fluorescence.
 24. Themethod of claim 20, wherein the biological molecule comprises a moleculeselected from the group consisting of a synthetic or naturally occurringprotein, a glycoprotein, a polypeptide, an amino acid, a nucleic acid, acarbohydrate, a lipid, a fatty acid, an aptamer, an antibody, and anycombination thereof. 25-26. (canceled)
 27. The method of claim 20,wherein the biological molecule is present on the surface of the cell,and adhering the bioconjugated chromophoric polymer dot to thebiological molecule comprises labeling the biological molecule with thebioconjugated chromophoric polymer dot.
 28. The method of claim 20,wherein the biological molecule is found inside the cell, and adheringthe bioconjugated chromophoric polymer dot to the biological moleculecomprises labeling the biological molecule with the bioconjugatedchromophoric polymer dot.
 29. The method of claim 20, wherein theamphiphilic molecule is attached to the chromophoric polymer by achemical association.
 30. The method of claim 20, wherein theamphiphilic molecule comprises an amphiphilic polymer, an amphiphiliccomb-like polymer, an amphiphilic copolymer, a polyalkylene glycol, alipid, a carbohydrate, or any combination thereof.
 31. The method ofclaim 20, wherein the chromophoric polymer dot further comprises: a corecomprising the chromophoric polymer and the hydrophobic moiety of theamphiphilic molecule; and a cap comprising the hydrophilic moiety of theamphiphilic molecule.
 32. The method of claim 31, wherein less than 100%of the cap is an organo silicate and the cap does not encapsulate thecore.
 33. The method of claim 20, wherein the hydrophobic moiety isphysically embedded in the chromophoric polymer by hydrophobicinteraction.
 34. The method of claim 20, wherein the amphiphilicmolecule comprises an amphiphilic polymer, an amphiphilic comb-likepolymer, an amphiphilic copolymer, a polyalkylene glycol, a lipid, acarbohydrate, or any combination thereof.
 35. The method of claim 34,wherein the amphiphilic polymer is a polystyrene-based comb-likepolymer, a poly(methyl methacrylate)-based comb-like polymer, or apolyethylene glycol.
 36. The method of claim 34, wherein the amphiphilicpolymer is a poly(styrene-co-maleic anhydride), a polyethyleneglycol-grafted polystyrene (PS-PEG), or a polystyrene grafted withethylene oxide functionalized with carboxyl groups (PS-PEG-COOH). 37.The method of claim 20, wherein the chromophoric polymer is a fluorenepolymer, a phenylene vinylene polymer, a phenylene polymer, a phenyleneethynylene polymer, a benzothiadiazole polymer, a thiophene polymer, acarbazole fluorene polymer, a boron-dipyrromethene-based polymer, anyderivative thereof, any copolymer thereof, or any combination thereof.38. The method of claim 20, wherein at least one of the one or morereactive functional groups is selected from the group consisting of acarboxyl, an amino, a mercapto, an azido, an alkynyl, a strainedalkynyl, an alkenyl, a strained alkenyl, a dienyl, a cyclooctynyl, analdehyde, a hydroxyl, a carbonyl, a sulfate, a sulfonate, a phosphate, acyanate, a succinimidyl ester, a substituted group thereof, and aderivative thereof.
 39. The method of claim 20, wherein the chromophoricpolymer is a semiconducting polymer.